Intracellular Levels Research Articles

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

Aristolochic acid induces acute kidney injury through ferroptosis.

Aristolochic acid (AA)-induced acute kidney injury (AKI) presents with progressive decline in renal function and rapid progression to end-stage renal disease. Among the multiple mechanisms identified in AKI, ferroptosis has been shown to be involved in various forms of AKI. But few studies have elucidated the role of ferroptosis in AA-induced AKI. In this study, we investigated the role of ferroptosis in AA-induced acute renal tubular injury in vivo and in vitro. Mice with acute aristolochic acid nephropathy showed increased malondialdehyde levels, aggravated lipid peroxidation, decreased superoxide dismutase activity, and glutathione depletion. The expression of glutathione peroxidase 4 was decreased and the expression of acyl-CoA synthetase long-chain family member 4 was increased. Inhibition of ferroptosis by ferrostatin-1 significantly improved the renal function, reduced histopathological lesions, partially alleviated lipid peroxidation, and restored the antioxidant capacity. In vitro studies also revealed that AA significantly reduced cell viability, induced reactive oxygen species production, increased intracellular iron level and decreased ferroptosis-related protein expression. Inhibition of ferroptosis significantly increased cell viability and attenuated AA-induced renal tubular epithelial cell injury. It is suggested that ferroptosis plays an important role in AA-induced acute tubular injury. And inhibition of ferroptosis may exert renoprotective effects possibly by preventing lipid peroxidation, restoring the antioxidant activity or regulating iron metabolism.

Novel methyltransferase G9a inhibitor induces ferroptosis in multiple myeloma through Nrf2/HO-1 pathway.

Multiple myeloma (MM) is a common malignant hematologic neoplasm, and the involvement of epigenetic modifications in its development and drug resistance has received widespread attention. Ferroptosis, a new ferroptosis-dependent programmed death mode, is closely associated with the development of MM. The novel methyltransferase inhibitor DCG066 has higher cell activity, but its mechanism of action in MM has not been clarified. Here, we found that DCG066 (5µM) inhibited the proliferation and induced ferroptosis in MM cells; the intracellular levels of ROS, iron, and MDA were significantly elevated, and the level of GSH was reduced after the treatment of DCG066; The protein expression levels of SLC7A11, GPX4, Nrf2 and HO-1 were significantly reduced, and these phenomena could be reversed by ferroptosis inhibitor Ferrostatin-1 (Fer-1) and Nrf2 activator Tert-butyl hydroquinone (TBHQ). Meanwhile, the protein expression levels of Keap1 was increased, and heat shock proteins (HSP70, HSP90 and HSPB1) were reduced after DCG066 treatment. In conclusion, this study confirmed that DCG066 inhibits MM proliferation and induces ferroptosis via the Nrf2/HO-1 pathway.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide

Ethnopharmacological relevanceAristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. Aim of the studyTo identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. Materials and methodsThe O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. ResultsAAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. ConclusionCollectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Attenuation of Nicotine Effects on A549 Lung Cancer Cells by Synthetic α7 nAChR Antagonists APS7-2 and APS8-2.

Nicotine binds to nicotinic acetylcholine receptors (nAChRs) that are overexpressed in different cancer cells, promoting tumor growth and resistance to chemotherapy. In this study, we aimed to investigate the potential of APS7-2 and APS8-2, synthetic analogs of a marine sponge toxin, to inhibit nicotine-mediated effects on A549 human lung cancer cells. Our electrophysiological measurements confirmed that APS7-2 and APS8-2 act as α7 nAChR antagonists. APS8-2 showed no cytotoxicity in A549 cells, while APS7-2 showed concentration-dependent cytotoxicity in A549 cells. The different cytotoxic responses of APS7-2 and APS8-2 emphasize the importance of the chemical structure in determining their cytotoxicity on cancer cells. Nicotine-mediated effects include increased cell viability and proliferation, elevated intracellular calcium levels, and reduced cisplatin-induced cytotoxicity and reactive oxygen species production (ROS) in A549 cells. These effects of nicotine were effectively attenuated by APS8-2, whereas APS7-2 was less effective. Our results suggest that APS8-2 is a promising new therapeutic agent in the chemotherapy of lung cancer.

Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells

Abstract Objectives Diabetic retinopathy (DR) is a retinal disease that arises from impaired glucose tolerance and leads to retinal microvascular leakages. Recent studies have indicated that DR pathogenesis is linked to dysfunctional retinal pigment epithelial (RPE) cells. Methods Investigating the potential interplay between endothelial cells (ECs) and RPE cells by treating ECs with high glucose (HG) and evaluating the function of cytokines released from ECs on the growth of RPE cells. Results The results revealed that high glucose-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) activated the NF-κB signaling pathway, increased intracellular levels of reactive oxygen species (ROS) and expression of caspase 3 while also elevating HUVECs delivery of cytokines such as VEGF, TNF-α, IL-6, and IL-1β. Conclusions As a result of our study, cytokines released from HG-treated HUVECs impede the growth of ARPE-19 in vitro, highlighting the importance of functional ECs for exploring the underlying mechanisms of vascular-associated retinal dysfunction. Inflammatory factors secreted from endothelial cells induced by high glucose impair human retinal pigment epithelial cells.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Furanocoumarins promote proteasomal degradation of viral HBx protein and down-regulate cccDNA transcription and replication of hepatitis B virus

Nucleot(s)ide analogues, the current antiviral treatments against chronic hepatitis B (CHB) infection, are non-curative due to their inability to eliminate covalently closed circular DNA (cccDNA) from the infected hepatocytes. Preclinical studies have shown that coumarin derivatives can effectively reduce the HBV DNA replication. We evaluated the antiviral efficacy of thirty new coumarin derivatives in cell culture models for studying HBV. Furanocoumarins Fc-20 and Fc-31 suppressed the levels of pre-genomic RNA as well as cccDNA, and reduced the secretion of virions, HBsAg and HBeAg. The antiviral efficacies of Fc-20 and Fc31 improved further when used in combination with the hepatitis B antiviral drug Entecavir. There was a marked reduction in the intracellular HBx level in the presence of these furanocoumarins due to proteasomal degradation resulting in the down-regulation of HBx-dependent viral genes. Importantly, both Fc-20 and Fc-31 were non-cytotoxic to cells even at high concentrations. Further, our molecular docking studies confirmed a moderate to high affinity interaction between furanocoumarins and viral HBx via residues Ala3, Arg26 and Lys140. These data suggest that furanocoumarins could be developed as a new therapeutic for CHB infection.

Abstract 5865: Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines

Abstract Background: The enzyme purine nucleoside phosphorylase (PNP) is involved in the purine salvage pathway and is present at high levels in lymphoid tissues. Children born with PNP deficiency are known to have significantly reduced T-cell counts but relatively normal B-cell immunity, suggesting PNP as one of the most promising therapeutic targets for T-cell malignancies. Forodesine (For, BCX-1777), a novel PNP inhibitor, was approved in Japan for the treatment of relapsed/refractory PTCL. To understand the cytotoxic effect of For and its mechanism of action, we established a For-resistant (For-R) leukemia cell line and explored the mechanism of resistance. Methods: We established two For-R T-lymphoblastic leukemia cell lines (CCRF-CEM and MOLT4) through repeated escalating exposure to For and 2’-deoxyguanosined(dGuo). Gene expression analysis was performed to identify the mechanisms of resistance. Results: For-R cells showed very high resistance compared to parental cells (IC50 value, CEM/CEM-R 4.2nM/>5x105nM, MOLT4/MOLT4-R 8nM/>5x105nM). When cross-resistance was examined in these For-R cells showed mild resistance to cytarabine. The expression of hENT1, a nucleoside transporter, was examined and found to be upregulated in both CEM-R and MOLT4-R cells, while the expression level of deoxycytidine kinase (dCK) was unchanged in both cells. Next, high performance liquid chromatography was used to assess the intracellular dGTP in each parental and its For-R cells. In the parental cells, intracellular dGTP levels increased promptly 4-6 hours after fosteredin administration, whereas in resistant cells, intracellular dGTP levels were higher than before fosteredin administration, and there was little change in dGTP levels after administration. Conclusion: The established For-R cell line acquired a high resistance to For and showed high intracellular dGTP levels. Imbalance in intracellular GTP levels may be involved in the induction of cell death by For. IC50 value of each cell lines Citation Format: Naoko Hosono, Kana Oiwa, Toshiki Tasaki, Rie Nishi, Luigi Scott, Owen A. O’Connor, Takahiro Yamauchi. Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines [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 5865.

Manganese dioxide nanoparticles provoke inflammatory damage in BV2 microglial cells via increasing reactive oxygen species to activate the p38 MAPK pathway.

With the widespread use of manganese dioxide nanoparticles (nano MnO2), health hazards have also emerged. The inflammatory damage of brain tissues could result from nano MnO2, in which the underlying mechanism is still unclear. During this study, we aimed to investigate the role of ROS-mediated p38 MAPK pathway in nano MnO2-induced inflammatory response in BV2 microglial cells. The inflammatory injury model was established by treating BV2 cells with 2.5, 5.0, and 10.0μg/mL nano MnO2 suspensions for 12 h. Then, the reactive oxygen species (ROS) scavenger (20nM N-acetylcysteine, NAC) and the p38 MAPK pathway inhibitor (10μM SB203580) were used to clarify the role of ROS and the p38 MAPK pathway in nano MnO2-induced inflammatory lesions in BV2 cells. The results indicated that nano MnO2 enhanced the expression of pro-inflammatory cytokines IL-1β and TNF-α, elevated intracellular ROS levels and activated the p38 MAPK pathway in BV2 cells. Controlling intracellular ROS levels with NAC inhibited p38 MAPK pathway activation and attenuated the inflammatory response induced by nano MnO2. Furthermore, inhibition of the p38 MAPK pathway with SB203580 led to a decrease in the production of inflammatory factors (IL-1β and TNF-α) in BV2 cells. In summary, nano MnO2 can induce inflammatory damage by increasing intracellular ROS levels and further activating the p38 MAPK pathway in BV2 microglial cells.

Abstract 3085: Cholesterol sulfotransferase SULT2B1b suppresses prostate tumor growth and regulates cholesterol metabolism in prostate cancer cells

Abstract Prostate cancer (PrCa) ranks as the second leading cause of cancer death in males in the United States. The 5-year survival rate drops drastically from 100% in patients with early-stage confined PrCa to 30% in those with advanced metastatic PrCa. Patients with advanced PrCa face limited long-term treatment options, often developing resistance to androgen-deprivation therapy and chemotherapy. Identifying and targeting pivotal molecules or pathways in advanced PrCa can substantially enhance patient survival. Our previous findings unveiled an inverse relationship between cholesterol sulfotransferase SULT2B1b and the severity of human PrCa. The highest levels of SULT2B1b were observed in the epithelium of normal prostates, with a progressive decrease as cancer advanced. The lowest levels were found in the most advanced metastatic samples. This study aims to determine whether SULT2B1b performs any active and critical functions in PrCa progression and assess whether it regulates cholesterol metabolism in PrCa cells. We generated SULT2B1b knockout (KO) clones from mouse PrCa cell line MyC-CaP and established inducible SULT2B1b overexpression lines from androgen receptor (AR)+ LNCaP and AR- PC3 human PrCa cells. These modified mouse and human cells were implanted into syngeneic FVB mice and immunodeficient NRG mice, respectively. MyC-CaP KO cells formed significantly larger tumors compared to parental cells, accompanied by reduced survival rates. In contrast, both LNCaP and PC3 tumors with induced SULT2B1b exhibited smaller sizes compared to control tumors, leading to improved survival rates. To investigate the impact of SULT2B1b on cholesterol metabolism and the underlying mechanisms behind SULT2B1b-regulated tumor growth, we examined genes related to lipoprotein signaling and cholesterol metabolism in MyC-CaP SULT2B1b KO cells using PCR arrays and qRT-PCR. Genes responsible for cholesterol uptake and synthesis (e.g. Ldlr, Cyp51, Sorl1, Pcsk9 and Insig1) were among the top upregulated genes, while genes controlling cholesterol efflux (e.g. Abca1) and esterification (e.g. Soat1 and Soat2) were downregulated in KO cells. Consistent with these gene expression patterns, intracellular cholesterol levels significantly increased in KO cells. Interestingly, although SULT2B1b did not affect cell proliferation under normal conditions, KO cells exhibited faster growth than parental cells when cultured in lipid-depleted or charcoal-stripped media, possibly due to altered intracellular cholesterol levels. In summary, these findings align with the expression pattern of SULT2B1b in human PrCa, demonstrating that SULT2B1b inhibits tumor growth, whereas its removal promotes tumor growth. This suggests SULT2B1b may serve as a critical tumor suppressor in PrCa. Its modulation of tumor growth may occur through the regulation of cholesterol metabolism. Citation Format: Jiang Yang, Dilinaer Wusiman, Timothy L. Ratliff. Cholesterol sulfotransferase SULT2B1b suppresses prostate tumor growth and regulates cholesterol metabolism in prostate cancer 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 3085.

Abstract 1892: KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors

Abstract Background: TfR1 is a transmembrane glycoprotein involved in iron uptake and regulation of cell growth. High TfR1 expression has been reported in different types of cancers, therefore recognized as a target molecule for cancer therapy. Despite the promise of potent efficacy of TfR1-targeted agents, there is a risk of side effects (i.e., severe anemia), since TfR1 is also highly expressed in certain normal cells (e.g., reticulocytes). KK2260 is a fully human tetravalent bispecific antibody (bsAb) based on REGULGENTTM technology targeting TfR1 and EGFR. This bsAb was designed for EGFR-expressing cell selective anti-cancer drug which could not be achieved by conventional monospecific Ab. In this study, we evaluated the mechanism of action (MOA), anti-cancer effect, and toxicity of KK2260. Methods: Binding activity of KK2260 to TfR1 and EGFR was evaluated by surface plasmon resonance method. Ligand blocking activity was evaluated by binding competition on esophageal squamous cell carcinoma (ESCC) OE21 cells. Growth inhibitory activity of KK2260 on cancer cells or hematopoietic progenitor cells was evaluated by proliferation inhibition assay. In some studies, excessive iron or anti-EGFR parent Ab was added. Effect on the cell surface target expression levels was evaluated by flow cytometry. The intracellular iron level was evaluated by cell imaging. Effect on EGF induced EGFR phosphorylation was evaluated by Western blotting. The in vivo efficacy of KK2260 was evaluated in cell line-derived and patient-derived xenograft (PDX) models of ESCC selected for high EGFR expression level. The toxicity of KK2260 was evaluated in cynomolgus monkeys. Results: KK2260 bound to TfR1 and EGFR with no ligand binding inhibition. KK2260 inhibited cell growth of moderate to high EGFR-expressing cancer cells, but not against EGFR-negative cancer cells or hematopoietic progenitor cells. KK2260 downregulated TfR1 expression on OE21 cells and the intracellular iron level decreased, but no effect was observed on EGFR phosphorylation. The growth inhibitory activity of KK2260 was abolished by adding excessive amounts of iron or reduced by inhibiting the binding to EGFR. Potent anti-tumor activity was observed in the ESCC PDX models with tumor regression. In the intermittent-dose toxicity study of cynomolgus monkeys, KK2260 was well-tolerated. Conclusions: KK2260 demonstrated potent anti-cancer effect both in vitro and in vivo. Intriguingly, KK2260 did not inhibit EGFR phosphorylation while EGFR-binding dependent TfR1 downregulation resulted in intracellular iron deficiency, which was considered as the MOA of KK2260. KK2260 was well tolerated in non-human primates. These preclinical data suggest the unique property of KK2260 is expected to provide significant benefit to patients with advanced ESCC and other EGFR-expressing tumors and Phase 1 study is currently on going in Japan (jRCT2031230372). Citation Format: Keisuke Mitamura, Hironori Otsuki, Keiko Nakahashi, Ayaka Kitagawa, Masayo Kamigaki, Yohei Kurio, Miho Inoue, Yasunori Uemura. KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors [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 1892.

Abstract 3067: SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway

Abstract SLC7A5/LAT1 is a cross-membrane transporter that functions in uptake of large neutral amino acids into cells. Many pathological studies have shown that LAT1 is highly expressed in cancer tissue from various origins such as lung, biliary tract, breast, prostate, bone, and their metastatic legions. High LAT1 expression has been recognized as a significant prognostic marker in various cancers. Our TCGA data analysis showed that the expression level of LAT1 is higher in TNBC tumors compared with normal breast tissues. The higher expression level of LAT1 is linked with poor overall and progression-free survival. Dysregulation of amino acid transporters lead to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer. However, it remains elusive whether and how LAT1 re-wires the cellular metabolic programs to promote TNBC tumor proliferation. In this study, we showed that inhibition of SLC7A5/LAT1 significantly reduced TNBC cell proliferation, viability, migration capability while promoting apoptosis. Mechanistically, we found significant positive correlations of LAT1 with PKM2 and LDHA in breast cancer tissues, which are key enzymes in mediating glycolysis and lactate fermentation or the Warburg Effect. Knock down LAT1 via siRNA decreased the expression levels of p-PKM2 and p-LDHA in TNBC cells. Tryptophan, a major substrate amino acid transported into cytosol by LAT1, is the precursor for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). The enzyme quinolinic acid phosphoribosyltransferase (QPRT) catalyzes the conversion from quinolinate, a product of tryptophan degradation, to NAD+. We showed LAT1 inhibition reduced the expression level of QPRT, cytosolic NAD+/NADH ratio as well as lactate production. Addition of L-Tryptophan increased the levels of p-PKM2 and p-LDHA while knockdown of LAT1 reversed tryptophan-mediated increase of PKM2 and LDHA activities, suggesting that LAT1 may regulate PKM2/LDHA through Trp/QPRT/NAD+ pathway for promoting the Warburg Effect. Additionally, we found upregulations of SLC7A5/LAT1, p-PKM2 and p-LDHA in doxorubicin-resistant cells and PDX mouse model compared with their corresponding controls. Collectively, these results provide evidence that overexpression of LAT1 in TNBC drives cancer progression and drug resistance by reinforcing the Warburg Effect, suggesting LAT1 as a promising therapeutic target for addressing TNBC. Our ongoing work will evaluate whether inhibiting LAT1 genetically or pharmacologically reduces xenograft tumor growth and enhances the sensitivities of TNBC resistant cells to chemotherapy in vivo. Our investigation will elucidate the underlying mechanisms involved in these processes. Citation Format: Margot Y. Fedoroff, Lei Zhao, Leili Saeednejad Zanjani, Jun He. SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway [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 3067.

Abstract 3189: A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity

Abstract Transmembrane 4 super family member 4(TM4SF4) protein has been shown to be involved in EMT-associated stemness through autocrine of insulin-like growth factor 1(IGF1) and osteopontin(OPN) in non-small cell lung cancer(NSCLC) cells. However, its potential as a therapeutic target has not been evaluated. In this study, anti-human TM4SF4 monoclonal antibodies(anti-hTM4SF4 mAbs) were prepared in mouse as part of a strategy for developing anticancer drug using humanized antibody. A synthetic peptide(15 mer, 126-140th AA) derived from the extracellular loop(ECL) 2 of human TM4SF4 exposed on outer cell surface was used as an immunization antigen. Among the selected anti-hTM4SF4 mAbs, ECL-2B7 and ECL-12A8 showed the best affinity for the antigen(Kd), approximately 2.66 nM and 8.34 nM, as determined by surface plasmon resonance analysis. Using mouse xenograft of NSCLC cell line, it was confirmed that ECL-2B7 was superior to ECL-12A8 in producing humanized antibody. ECL-2B7 inhibited EMT-associated stemness of TM4SF4 positive cancer cells by blocking cellular signaling events such as IGF1Rβ and CD44 and their downstream signals, PI3K/AKT/GSK3β/NF-κB and JAK2/STAT3 pathways. ECL-2B7 also enhanced antitumor activity by downregulating cellular and exosomal level of PD-L1 and B7-H4, immune-checkpoint ligands that elicit immunosuppression of T cell, and by mediating antibody-dependent cellular cytotoxicity. Treatment with OPN- and IGF1-neutralizing antibodies suppressed intracellular PD-L1 and B7-H4 level. It means that the regulation of PD-L1 and B7-H4 levels by TM4SF4 is closely related to the autocrine action of OPN and IGF1 induced by TM4SF4. Using patient-derived xenograft model, it was confirmed that the ECL-2B7 has excellent antitumor activity that effectively inhibits tumor progress. These results strongly suggest that it is necessary to construct humanized antibody based on ECL-2B7 to confirm its potential as a novel anticancer drug. Citation Format: Rae Kwon Kim, Chang-Kyu Heo, Yeon-jee Kahm, Uhee Jung, Byung-Chul Shin, Won-Yong Kim, Bum-Jin Kim, Sung-Chul Kim, Chun-jeih Ryu, Eun-Wie Cho, In-Gyu Kim. A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity [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 3189.

Abstract 3227: Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM

Abstract Background: Homozygous deletion of MTAP is one of the most frequent genetic alterations in various solid tumors including glioblastoma (GBM), mesothelioma, pancreatic cancer and lung cancer. While MTAP-deleted cancers are associated with a poor prognosis, there are no approved drugs for the treatment of patients with MTAP deficiency. MAT2A has emerged as a potential target for selective anticancer effects in MTAP-deleted cancers. However, development of brain-penetrating MAT2A inhibitors are yet to be reported in preclinical or clinical stages despite of high MTAP deletion fraction in GBM and brain metastatic cancers. Here, we present highly potent and selective MAT2A inhibitors capable of brain penetration for MTAP deleted cancer therapy. Methods: A MAT2A biochemical enzyme assay was performed with phosphonate colorimetric kit. To evaluate in vitro and in vivo activities of compounds, S-adenosylmethionine (SAM) and symmetrical dimethyl arginine (SDMA) levels were measured using LC-MS/MS and high-content screening systems. Cell growth inhibition assay was evaluated to confirm the selectivity in isogenic pairs of HCT116 WT and MTAP-deleted cells. In vivo efficacy was conducted using the MTAP-deleted HCT116 xenograft. Results: To develop novel brain penetrating scaffolds, we focused on interaction of hydrogen bond acceptor in the scaffolds with arginine residue Arg313 in MAT2A protein and the improvement of physicochemical properties, including logP and logBB. A series of MAT2A inhibitors significantly inhibited MAT2A enzymatic activity, resulting in the reduction of intracellular SAM level, the direct product of MAT2A. These compounds showed highly potent inhibition of SDMA and cell proliferation in MTAP-deleted cells, while displaying minimal effect on MTAP-WT cells, showing >100-fold selectivity towards MTAP-deletion over MTAP-WT. Furthermore, anti-proliferative activity of MAT2A inhibitors were observed in MTAP-deleted cancer cells across various cancer types, including GBM, breast cancer, and non-small cell lung cancer. Remarkably, mouse pharmacokinetic study revealed high level of plasma and brain exposure with outstanding brain-to-plasma ratio (> 0.75). Robust tumor growth inhibition as well as reduction in tumor SAM levels were observed as monotherapy in an MTAP-deleted HCT116 xenograft model. Conclusion: We have identified novel brain-penetrating MAT2A inhibitors displaying encouraging in vitro and in vivo pharmacological profiles, indicating the potential use of these compounds as therapeutic agents for MTAP deleted brain metastatic cancer and GBM. Citation Format: Soyoung Ki, Jinhee Kim, Jungtae Na, Ilkyoo Koh, Hyunho Cho, Ho Yeon Lee, Hoiyun Jung, Gyeonghi Cho, Mijin Moon, Yongje Shin, Sook-Kyung Park. Developing brain-penetrating MAT2A inhibitors for MTAP-deleted brain metastatic cancer and GBM [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 3227.

SBFI26 induces triple-negative breast cancer cells ferroptosis via lipid peroxidation.

SBFI26, an inhibitor of FABP5, has been shown to suppress the proliferation and metastasis of tumour cells. However, the underlying mechanism by which SBFI26 induces ferroptosis in breast cancer cells remains largely unknown. Three breast cancer cell lines were treated with SBFI26 and CCK-8 assessed cytotoxicity. Transcriptome was performed on the Illumina platform and verified by qPCR. Western blot evaluated protein levels. Malondialdehyde (MDA), total superoxide dismutase (T-SOD), Fe, glutathione (GSH) and oxidized glutathione (GSSG) were measured. SBFI26 induced cell death time- and dose-dependent, with a more significant inhibitory effect on MDA-MB-231 cells. Fer-1, GSH and Vitamin C attenuated the effects but not erastin. RNA-Seq analysis revealed that SBFI26 treatment significantly enriched differentially expressed genes related to ferroptosis. Furthermore, SBFI26 increased intracellular MDA, iron ion, and GSSG levels while decreasing T-SOD, total glutathione (T-GSH), and GSH levels.SBFI26 dose-dependently up-regulates the expression of HMOX1 and ALOX12 at both gene and protein levels, promoting ferroptosis. Similarly, it significantly increases the expression of SAT1, ALOX5, ALOX15, ALOXE3 and CHAC1 that, promoting ferroptosis while downregulating the NFE2L2 gene and protein that inhibit ferroptosis. SBFI26 leads to cellular accumulation of fatty acids, which triggers excess ferrous ions and subsequent lipid peroxidation for inducing ferroptosis.

Abstract 2978: Extracellular ATP induced senescence in human lung cancer cells

Abstract In tumors, extracellular ATP (eATP) concentrations are 1,000 to 10,000 times higher than is found in normal tissues and eATP is up taken through macropinocytosis leading to a large increase in intracellular ATP levels. eATP is also linked to the TGF-β signaling pathway as exocytosis of ATP is induced that is then used in an autocrine fashion activating the P2 × 7 purinergic receptor1. Through these mechanisms, eATP has also been recently shown to induce EMT, CSC formation, and drug resistance1. One of the mechanisms through which these processes may be induced is a senescent cell precursor2. Senescence is a generally irreversible epigenetically controlled process characterized by a complete exit from the cell cycle in response to either DNA damage, stressful conditions, or aging. Recent mounting evidence has shown that senescence can be a cancer promoting mechanism by entering this state in response to stress such as during chemotherapy, but they can utilize it to enhance their malignant capabilities, including the ability to escape their senescent state with heightened stem cell characteristics, which can lead to a higher chance of relapse after treatment2. Senescent cells have recently been named a hallmark of cancer3. This study shows the time dependent induction of a senescent like state in human lung cancer A549 cells after only 2 hours of treatment with 0.5mM eATP, the concentration found in the tumor microenvironment, through analysis of transcriptional markers using total polyA RNAseq as well as chromogenic and flow cytometric assays for senescence associated beta-galactosidase activity. Additionally, inhibitors of macropinocytosis and purinergic receptors have been used to gain insight into the mechanisms of action that this pathway takes. This demonstrates a novel use of ATP within the tumor microenvironment for aiding in cancer progression and drug resistance. After EMT, CSC formation, and drug resistance, senescence is added to the long list of eATP mediated cancer promoting processes. 1. Song J, Qian Y, Evers M, Nielsen CM, Chen X. Cancer Stem Cell Formation Induced and Regulated by Extracellular ATP and Stanniocalcin-1 in Human Lung Cancer Cells and Tumors. IJMS. 2022;23(23):14770. doi:10.3390/ijms232314770 2. Zhang D, Monteiro MJ, Liu J, Gu W. Mechanisms of cancer stem cell senescence: Current understanding and future perspectives. Clin Exp Pharma Physio. 2021;48(9):1185-1202. doi:10.1111/1440-1681.13528 3. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discovery. 2022;12(1):31-46. doi:10.1158/2159-8290.CD-21-1059 Citation Format: Ryan A. Ward, Arabella Hunt, Nora Anderson, Xiaozhuo Chen. Extracellular ATP induced senescence in human lung cancer 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 2978.

Abstract 3068: CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming

Abstract Background & Aim: In the tumor microenvironment, the adipocytes enhance the invasiveness of breast cancer (BC) by remodeling the lipid metabolism, especially in triple-negative breast cancer (TNBC). The cell adhesion molecule CD146 was highly expressed in TNBC and predicted poor distant metastasis free survival (DMFS) in patients. Meanwhile, CD146 was known to alter the obesity process by regulating the metabolic level of the body. In this study, we aimed to explore the molecular mechanism of CD146 in tumor metastasis mediating by lipid metabolic reprogramming between adipocytes and TNBC cells. Methods: CD146 knockout (KO) or overexpression cell models were constructed by CRISPR/Cas9 or lentivirus. Cells labeled with luciferase were injected in tail vein of Balb/c-nude mice and monitored lung metastasis by IVIS Kinetic system in vivo. Transwell and wound healing assays were analyzed cell aggressiveness in vitro. We synthesized anti-CD146 nanobodies, which significantly degraded CD146 protein and inhibitd its function. RNA-seq and Lipidomics in CD146 knockout models were performed to analyze the role of CD146 in TNBC cell metabolism. Immunofluorescence was used to detect the intracellular content of Lipid Droplet (LD) and protein localization. Finally, we used oleic acid (OA) and adipocyte-conditioned medium (CM) to mimic the tumor microenvironment. Results: CD146 was highly expressed in TNBC and predicted poor DMFS survival. Inhibition of CD146 suppressed tumor aggressiveness and lung metastasis in vitro and in vivo. According to the bioinformatics and RNA-Seq analysis, we showed that CD146 was intimately involved in the lipid metabolic pathway, particularly fatty acid metabolism, and revealed that inhibiting CD146 in TNBC elevated the intracellular triglyceride level through lipidomic assay. Meanwhile, knockout of CD146 promoted LD accumulation and decreased fatty acid oxidation (FAO) and mitochondrial ATP levels. In molecular mechanism, we found that adipocytes secrete high level of IL6 and fatty acid, which activated JAK/STAT3 signaling and FAO mediating by CD146 in TNBC cells. Blocking CD146 using its nanobody inhibited IL6/JAK/STAT3 activities and ACADM, leading to impairment of FAO and lung metastasis suppression in vitro and in vivo. Conclusions: In this study, we demonstrate the crucial role of CD146 in the crosstalk between tumor cells and adipocytes in the tumor microenvironment. CD146 promotes the utilization of fatty acids through fatty acid oxidation and activates the downstream inflammatory pathway response with adipocyte-secreted IL-6, thereby driving BC aggressiveness. Our study has important implications for the development of TNBC therapeutics to block CD146-driven FAO process, inflammatory response and tumor metastasis. Citation Format: Hexing Sun, Kaiyuan Huang, Bingmei Sun, Jiajia Lin, Ruiqian Yang, Jiawei Li, Shimeng Wang, De Zeng, Haoyu Lin, Yuanke Liang. CD146mediates intimate crosstalk between adipocytes and triple-negative breast cancer cells and drives tumor metastasis through lipid metabolic reprogramming [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 3068.

Protective Effects and Mechanisms of Esculetin against H2O2-Induced Oxidative Stress, Apoptosis, and Pyroptosis in Human Hepatoma HepG2 Cells.

Oxidative stress plays a crucial role in the pathogenesis of many diseases. Esculetin is a natural coumarin compound with good antioxidant and anti-inflammatory properties. However, whether esculetin can protect HepG2 cells through inhibiting H2O2-induced apoptosis and pyroptosis is still ambiguous. Therefore, this study aimed to investigate the protective effects and mechanisms of esculetin against oxidative stress-induced cell damage in HepG2 cells. The results of this study demonstrate that pretreatment with esculetin could significantly improve the decrease in cell viability induced by H2O2 and reduce intracellular ROS levels. Esculetin not only apparently reduced the apoptotic rates and prevented MMP loss, but also markedly decreased cleaved-Caspase-3, cleaved-PARP, pro-apoptotic protein (Bax), and MMP-related protein (Cyt-c) expression, and increased anti-apoptotic protein (Bcl-2) expression in H2O2-induced HepG2 cells. Meanwhile, esculetin also remarkably reduced the level of LDH and decreased the expression of the pyroptosis-related proteins NLRP3, cleaved-Caspase-1, Il-1β, and GSDMD-N. Furthermore, esculetin pretreatment evidently downregulated the protein expression of p-JNK, p-c-Fos, and p-c-Jun. Additionally, anisomycin, a specific activator of JNK, blocked the protection of esculetin against H2O2-induced HepG2 cells apoptosis and pyroptosis. In conclusion, esculetin can protect HepG2 cells against H2O2-induced oxidative stress, apoptosis, and pyroptosis via inhibiting the JNK signaling pathway. These findings indicate that esculetin has the potential to be used as an antioxidant that improves oxidative stress-related diseases.