Nude Mouse Xenograft Model Research Articles

Combination of betulinic acid and EGFR-TKIs exerts synergistic anti-tumor effects against wild-type EGFR NSCLC by inducing autophagy-related cell death via EGFR signaling pathway

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of lung cancer patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type EGFR tumors has been shown to be marginal. Methods that can sensitize EGFR-TKIs to EGFR wild-type NSCLC remain rare. Hence, we determined whether combination treatment can maximize the therapeutic efficacy of EGFR-TKIs.MethodsWe established a focused drug screening system to investigate candidates for overcoming the intrinsic resistance of wild-type EGFR NSCLC to EGFR-TKIs. Molecular docking assays and western blotting were used to identify the binding mode and blocking effect of the candidate compounds. Proliferation assays, analyses of drug interactions, colony formation assays, flow cytometry and nude mice xenograft models were used to determine the effects and investigate the molecular mechanism of the combination treatment.ResultsBetulinic acid (BA) is effective at targeting EGFR and synergizes with EGFR-TKIs (gefitinib and osimertinib) preferentially against wild-type EGFR. BA showed inhibitory activity due to its interaction with the ATP-binding pocket of EGFR and dramatically enhanced the suppressive effects of EGFR-TKIs by blocking EGFR and modulating the EGFR-ATK-mTOR axis. Mechanistic studies revealed that the combination strategy activated EGFR-induced autophagic cell death and that the EGFR-AKT-mTOR signaling pathway was essential for completing autophagy and cell cycle arrest. Activation of the mTOR pathway or blockade of autophagy by specific chemical agents markedly attenuated the effect of cell cycle arrest. In vivo administration of the combination treatment caused marked tumor regression in the A549 xenografts.ConclusionsBA is a potential wild-type EGFR inhibitor that plays a critical role in sensitizing EGFR-TKI activity. BA combined with an EGFR-TKI effectively suppressed the proliferation and survival of intrinsically resistant lung cancer cells via the inhibition of EGFR as well as the induction of autophagy-related cell death, indicating that BA combined with an EGFR-TKI may be a potential therapeutic strategy for overcoming the primary resistance of wild-type EGFR-positive lung cancers.Graphical abstract

ZNT5-6 and ZNT7 play an integral role in protein N-glycosylation by supplying Zn2+ to Golgi α-mannosidase II

The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.

High-Affinity NIR-Fluorescent Inhibitors for Tumor Imaging via Carbonic Anhydrase IX.

Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity Kd value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.

β-Elemene Reverses Gefitinib Resistance in NSCLC Cells by Inhibiting lncRNA H19-Mediated Autophagy.

Lung cancer is a leading cause of mortality worldwide, especially among Asian patients with non-small cell lung cancer (NSCLC) who have epidermal growth factor receptor (EGFR) mutations. Initially, first-generation EGFR tyrosine kinase inhibitors (TKIs) are commonly administered as the primary treatment option; however, encountering resistance to these medications poses a significant obstacle. Hence, it has become crucial to address initial resistance and ensure continued effectiveness. Recent research has focused on the role of long noncoding RNAs (lncRNAs) in tumor drug resistance, especially lncRNA H19. β-elemene, derived from Curcuma aromatic Salisb., has shown strong anti-tumor effects. However, the relationship between β-elemene, lncRNA H19, and gefitinib resistance in NSCLC is unclear. This study aims to investigate whether β-elemene can enhance the sensitivity of gefitinib-resistant NSCLC cells to gefitinib and to elucidate its mechanism of action. The impact of gefitinib and β-elemene on cell viability was evaluated using the cell counting kit-8 (CCK8) assay. Furthermore, western blotting and qRT-PCR analysis were employed to determine the expression levels of autophagy-related proteins and genes, respectively. The influence on cellular proliferation was gauged through a colony-formation assay, and apoptosis induction was quantified via flow cytometry. Additionally, the tumorigenic potential in vivo was assessed using a xenograft model in nude mice. The expression levels of LC3B, EGFR, and Rab7 proteins were examined through immunofluorescence. Our findings elucidate that the resistance to gefitinib is intricately linked with the dysregulation of autophagy and the overexpression of lncRNA H19. The synergistic administration of β-elemene and gefitinib markedly attenuated the proliferative capacity of resistant cells, expedited apoptotic processes, and inhibited the in vivo proliferation of lung cancer. Notably, β-elemene profoundly diminished the expression of lncRNA H19 and curtailed autophagic activity in resistant cells, thereby bolstering their responsiveness to gefitinib. Moreover, β-elemene disrupted the Rab7-facilitated degradation pathway of EGFR, facilitating its repositioning to the plasma membrane. β-elemene emerges as a promising auxiliary therapeutic for circumventing gefitinib resistance in NSCLC, potentially through the regulation of lncRNA H19-mediated autophagy. The participation of Rab7 in this dynamic unveils novel insights into the resistance mechanisms operative in lung cancer, paving the way for future therapeutic innovations.

KIF22 promotes multiple myeloma progression by regulating the CDC25C/CDK1/cyclinB1 pathway

PurposeMultiple myeloma (MM) is an incurable hematological malignancy characterized by clonal proliferation of malignant plasma B cells in bone marrow, and its pathogenesis remains unknown. The aim of this study was to determine the role of kinesin family member 22 (KIF22) in MM and elucidate its molecular mechanism.MethodsThe expression of KIF22 was detected in MM patients based upon the public datasets and clinical samples. Then, in vitro assays were performed to investigate the biological function of KIF22 in MM cell lines, and subcutaneous xenograft models in nude mice were conducted in vivo. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay were used to determine the mechanism of KIF22-mediated regulation.ResultsThe results demonstrated that the expression of KIF22 in MM patients was associated with several clinical features, including gender (P = 0.016), LDH (P < 0.001), β2-MG (P = 0.003), percentage of tumor cells (BM) (P = 0.002) and poor prognosis (P < 0.0001). Furthermore, changing the expression of KIF22 mainly influenced the cell proliferation in vitro and tumor growth in vivo, and caused G2/M phase cell cycle dysfunction. Mechanically, KIF22 directly transcriptionally regulated cell division cycle 25C (CDC25C) by binding its promoter and indirectly influenced CDC25C expression by regulating the ERK pathway. KIF22 also regulated CDC25C/CDK1/cyclinB1 pathway.ConclusionKIF22 could promote cell proliferation and cell cycle progression by transcriptionally regulating CDC25C and its downstream CDC25C/CDK1/cyclinB1 pathway to facilitate MM progression, which might be a potential therapeutic target in MM.

Downregulation of lncRNA MIR17HG reduced tumorigenicity and Treg-mediated immune escape of non-small-cell lung cancer cells through targeting the miR-17-5p/RUNX3 axis.

Long noncoding RNA MIR17HG was involved with the progression of non-small-cell lung cancer (NSCLC), but specific mechanisms of MIR17HG-mediated immune escape of NSCLC cells were still unknown. The present study investigated the function of MIR17HG on regulatory T cell (Treg)-mediated immune escape and the underlying mechanisms in NSCLC. Expression of MIR17HG and miR-17-5p in NSCLC tissue samples were detected using quantitative real-time PCR (qRT-PCR). A549 and H1299 cells were transfected with sh-MIR17HG, miR-17-5p inhibitor, or sh-MIR17HG + miR-17-5p inhibitor, followed by cocultured with Tregs. Cell proliferation was measured using 5-ethynyl-20-deoxyuridine (Edu) staining assay and cell counting kit-8 (CCK-8) assay. Flow cytometry was used for determining positive numbers of FOXP3+CD4+/CD25+/CD8+ Tregs. Through subcutaneous injection with transfected A549 cells, a xenograft nude mouse model was established. Weights and volumes of xenograft tumors were evaluated. Additionally, the expressions of immune-related factors includingtransforming growth factor beta (TGF-β), vascular endothelial growth factor A (VEGF-A), interleukin-10 (IL-10), IL-4, and interferon-gamma (IFN-γ) in cultured cells, were evaluated by enzyme-linked immunosorbent assay and western blot analysis. Then, miR-17-5p was decreased and MIR17HG was enhanced in both NSCLC tissues and cell lines. MIR17HG knockdown significantly suppressed cell proliferation, tumorigenicity, and immune capacity of Tregs in A549 and H1299 cells, whereas sh-MIR17HG significantly reduced expression levels of VEGF-A, TGF-β, IL-4, and IL-10 but promoted the IFN-γ level in vitro and in vivo. Moreover, downregulation of miR-17-5p significantly reversed the effects of sh-MIR17HG. Additionally, we identified that runt- related transcription factor 3 (RUNX3) was a target of miR-17-5p, and sh-MIR17HG and miR-17-5p mimics downregulated RUNX3 expression. In conclusion, downregulation of MIR17HG suppresses tumorigenicity and Treg-mediated immune escape in NSCLC through downregulating the miR-17-5p/RUNX3 axis, indicating that this axis contains potential biomarkers for NSCLC.

Anlotinib Inhibiting Mantle Cell Lymphoma Proliferation and Inducing Apoptosis through PI3K/AKT/mTOR Pathway.

This study investigates the inhibitory mechanism of anlotinib on human Mantle Cell Lymphoma (MCL) cells through in vitro and in vivo experiments. In vitro cellular experiments validate the effects of anlotinib on MCL cell proliferation and apoptosis. Moreover, a subcutaneous xenograft nude mice model of Mino MCL cells was established to assess the anti-tumour effect and tumour microenvironment regulation of anlotinib in vivo. The results indicate that MCL cell proliferation was significantly inhibited upon anlotinib exposure. The alterations in the expression of apoptosis-related proteins further confirm that anlotinib can induce apoptosis in MCL cells. Additionally, anlotinib significantly reduced the PI3K/Akt/mTOR phosphorylation level in MCL cells. The administration of a PI3K phosphorylation agonist, 740YP, could reverse the inhibitory effect of anlotinib on MCL. In the xenograft mouse model using Mino MCL cells, anlotinib treatment led to a gradual reduction in body weight and a significant increase in survival time compared to the control group. Additionally, anlotinib attenuated PD-1 expression and elevated inflammatory factors, CD4, and CD8 levels in tumour tissues. Anlotinib effectively inhibits proliferation and induces apoptosis in MCL both in vitro and in vivo. This inhibition is likely linked to suppressing phosphorylation in the PI3K/Akt/mTOR pathway.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt

Ethnopharmacological relevanceLicorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. Aim of the studyThis study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. Materials and methodsSW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. ResultsWighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a “closed” conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. ConclusionWighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Activation of CTU2 expression by LXR promotes the development of hepatocellular carcinoma.

Cytosolic thiouridylase 2 (CTU2) is an enzyme modifying transfer RNAs post-transcriptionally, which has been implicated in breast cancer and melanoma development. And we found CTU2 participated in hepatocellular carcinoma (HCC) progression here. HepG2 cells as well as xenograft nude mice model were employed to investigate the role of CTU2 in HCC development in vitro and in vivo respectively. Further, we defined CTU2 as a Liver X receptor (LXR) targeted gene, with a typical LXR element in the CTU2 promoter. CTU2 expression was activated by LXR agonist and depressed by LXR knockout. Interestingly, we also found CTU2 took part in lipogenesis by directly enhancing the synthesis of lipogenic proteins, which provided a novel mechanism for LXR regulating lipid synthesis. Meanwhile, lipogenesis was active during cell proliferation, particularly in tumor cells. Reduction of CTU2 expression was related to reduced tumor burden and synergized anti-tumor effect of LXR ligands by inducing tumor cell apoptosis and inhibiting cell proliferation. Taken together, our study identified CTU2 as an LXR target gene. Inhibition of CTU2 expression could enhance the anti-tumor effect of LXR ligand in HCC, identifying CTU2 as a promising target for HCC treatment and providing a novel strategy for the application of LXR agonists in anti-tumor effect.

SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1–involved EMT in hepatocellular carcinoma

BackgroundThe protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression.MethodsThe modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient’s overall survival versus AnxA6 expression level was evaluated by the Kaplan–Meier method.ResultsLys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients.ConclusionsAnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

α-Hederin induces paraptosis by targeting GPCRs to activate Ca2+/MAPK signaling pathway in colorectal cancer.

Non-apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α-hederin (α-hed) induces caspase-independent paraptotic modes of cell death. The present study is aimed to investigate the role of α-hed induces paraptosis and the associated mechanism of it. The cell proliferation was detected by CCK-8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α-hed. Molecular docking methods were used to evaluate binding abilities of α-hed with targets. The expressions of genes and proteins were analyzed by RT-qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects invivo. α-hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg-2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α-hed. Besides, both Swiss prediction and molecular docking showed that the structure of α-hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)-β3/ inositol 1,4,5-trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α-hed. After blocking the GPCR signaling, α-hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α-hed activated MAPK cascade by elevating Ca2+ flux. Since non-apoptotic cell death is presently emerging as a potential direction to overcome chemo-drug resistance, we then found α-hed also induced paraptosis in 5-fluorouracil-resistant (5-FU-R) CRC cells, and it reduced the growth of 5-FU-R CRC xenografts. Collectively, our findings proved α-hed as a promising candidate for inducing non-apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic-based chemo-resistance in CRC.

PSMA2 promotes glioma proliferation and migration via EMT

BackgroundGliomas advance rapidly and are associated with a poor prognosis. Epithelial-mesenchymal transition (EMT) accelerates the progression of gliomas, exerting a pivotal role in glioma development. Proteasome subunit alpha type-2 (PSMA2) exhibits high expression levels in gliomas. however, its specific involvement in glioma progression and its correlation with EMT remain elusive. This study aims to elucidate the role of PSMA2 in glioma progression and its potential association with EMT. MethodsOnline tools were employed to analyze the expression patterns and survival curves of PSMA2 in gliomas. The relationship between PSMA2 and various characteristics of glioma patients was investigated using data from the TCGA and CGGA databases. In vitro, cell proliferation and migration were assessed through CCK-8, colony formation, and transwell assays. Furthermore, a tumor xenograft model in nude mice was established to evaluate in vivo tumorigenesis. Protein binding to PSMA2 was scrutinized using co-immunoprecipitation MS (co-IP MS). The potential biological functions and molecular pathways associated with PSMA2 were explored through GO analysis and KEGG analysis, and the correlation between PSMA2 and EMT was validated through correlation analysis and Western blot experiments. ResultsBioinformatics analysis revealed a significant upregulation of PSMA2 across various cancers, with particularly heightened expression in gliomas. Moreover, elevated PSMA2 levels were correlated with advanced tumor stages and diminished survival rates among glioma patients. Inhibition of PSMA2 demonstrated a pronounced suppressive effect on glioma cell proliferation, both in vitro and in vivo. Knockdown of PSMA2 also impeded the migratory capacity of glioma cells. GO and KEGG enrichment analyses indicated that PSMA2-binding proteins (identified through Co-IP-MS) were associated with cell adhesion molecule binding and cadherin binding. Western blot results further confirmed the role of PSMA2 in promoting epithelial-mesenchymal transition (EMT) in glioma cells. ConclusionOur study provides evidence supporting the role of PSMA2 as a regulatory factor in EMT and suggests its potential as a prognostic biomarker for glioma progression.

Trastuzumab-conjugated liposomes for co-delivery of paclitaxel and anti-abcb1 siRNA in HER2-positive breast cancer: In vitro and in vivo evaluations

Chemotherapeutic drugs often face challenges such as non-specific binding, undesired toxicity, and limited blood circulation, leading to reduced therapeutic efficacy. This study introduces HER2-specific targeted nanoparticles designed by synthesizing cationic liposomes. These liposomes encapsulate paclitaxel and ABCB1-siRNA in their core and feature a therapeutic monoclonal antibody, trastuzumab, on the surface. This design aims for precise targeting and synergistic treatment of HER2-positive breast cancer cells in both in vitro and in vivo models. The optimized trastuzumab-conjugated liposomes exhibited a particle size of 229 ± 4 nm and a zeta potential of 43.46 ± 0.61 mV. Their spherical morphology was confirmed using Scanning Electron Microscopy (SEM). These liposomes demonstrated enhanced drug retention in in vitro release studies. Furthermore, trastuzumab-conjugated liposomes displayed markedly higher cellular uptake than their non-trastuzumab-conjugated counterparts. Their anticancer efficacy in BT-474 cells significantly surpassed that of non-targeted liposomes and unencapsulated paclitaxel. In xenograft Nude mice models derived from the BT-474 cell line, trastuzumab-conjugated liposomes showed superior tumor distribution and preclinical effectiveness compared to both non-targeted liposomes and plain paclitaxel.

Schizandrin A enhances the sensitivity of gastric cancer cells to 5-FU by promoting ferroptosis.

Schizandrin A (Sch A) exert anticancer and multidrug resistance-reversing effects in a variety of tumors, but its effect on 5-fluorouracil (5-Fu) in gastric cancer (GC) cells remains unclear. The aim of the present study was to examine the resistance-reversing effect of Schizandrin A and assess its mechanisms in 5-Fu-resistant GC cells.5-Fu-sensitive GC cells were treated with 5-Fu and 5-Fu-resistant GC cells AGS/5-Fu and SGC7901/5-Fu were were established. These cells were stimulated with Schizandrin A alone or co-treated with 5-Fu and their effect on tumor cell growth, proliferation, migration, invasion and ferroptosis-related metabolism were investigated both in vitro and in vivo. A number of additional experiments were conducted in an attempt to elucidate the molecular mechanism of increased ferroptosis. The results of our study suggest that Schizandrin A in combination with 5-Fu might be useful in treating GC by reverse drug resistance. It was shown that Schizandrin A coadministration suppressed metastasis and chemotherapy resistance in 5-Fu-resistant GC cells through facilitating the onset of ferroptosis, which is an iron-dependent form of cell death, which was further demonstrated in a xenograft nude mouse model. Mechanistically, Schizandrin A co-administration synergistically increased the expression of transferin receptor, thus iron accumulates within cells, leading to lipid peroxidation, which ultimately results in 5-Fu-resistant GC cells death. The results of this study have provided a novel strategy for increasing GC chemosensitivity, indicating Schizandrin A as a novel ferroptosis regulator. Mechanistically, ferroptosis is induced by Schizandrin A coadministration via increasing transferrin receptor expression.

Abstract 4676: 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (MT2) is a novel paclitaxel chemosensitizer to inhibit growth in Taxol non-resistant and Taxol resistant cancers

Abstract 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (MT2) is a single molecule isolated from a traditional Chinese Medicinal herb Marsdenia tenacissima (MT). Recently, our group has isolated a few single molecules from the MT herb, they have shown significant anti-tumor activity in vitro and MT2 has the highest potential among all. We have performed MTT viability assay and clonogenic assay to demonstrate that MT2-alone is non-cytotoxic, but the cytotoxicity of the paclitaxel+MT2 combination treatment was more significant than that of paclitaxel, a microtubule stabilizing chemotherapeutic agent. The underlying mechanism of such growth inhibition is contributed by the down-regulation of the pro-survival signaling and up-regulation of the apoptotic signaling such as an increase in cleaved caspase 3 and decrease in anti-apoptotic Bcl-2 proteins when cancer cells are co-treated with MT2+paclitaxel. More importantly, intraperitoneal administration of the combination treatment 5 times of MT2+paclitaxel (30mg/kg, 8mg/kg) has a more significant anti-tumor effect than the paclitaxel-alone (8mg/kg) or MT2-alone (30mg/kg) treatments using the wild-type hepatic HepG2 xenograft model in nude mice. Besides the wild-type HepG2 cancer cells, we have further demonstrated that MT2 is able to resensitize paclitaxel-resistant HepG2 cancer cells by inhibiting the activity of p-glycoprotein (Pgp), a key drug efflux transporter in chemotherapy resistance cancer cells. Also, the combination treatment has shown a significant anti-tumor effect in HepG2/DOX Pgp-overexpressed xenograft in nude mice. Taken together, MT2 is a novel chemosensitiser in combination use with paclitaxel to treat cancer. It enhances cancer apoptosis by suppressing the anti-apoptotic and survival signaling pathways in cancer cells. Citation Format: Ka Ming Cheng, Gui yun Wu, Kowk Kuen Cheung, Xiao-Ling Shen, Ying-Jie Hu, Ki Lui. 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (MT2) is a novel paclitaxel chemosensitizer to inhibit growth in Taxol non-resistant and Taxol resistant cancers [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 4676.

Abstract 5595: Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker

Abstract Background: In our previous study, based on LC-MS/MS analysis, the salivary Cornulin, a relatively less explored protein, was found to be ~10-fold downregulated in Head and Neck Squamous Cell Carcinoma (HNSCC) patients. However, the exact role of Cornulin in the pathophysiology of HNSCC is not reported. Here, we investigated its role in the pathophysiology of HNSCC and elucidated its potential as a prognostic marker as well as its antitumour effect. Methods: Sandwich ELISA and immunohistochemistry were performed to estimate the salivary and tissue levels of Cornulin, respectively, in HNSCC patients, along with an analysis of the effects of Cornulin levels on disease outcome. To investigate the antitumor effects ,its levels were upregulated through lentiviral transduction in low expressing Cal27, FaDu cell lines. Subsequently, various assays were conducted to examine the antitumor effects of Cornulin upregulation. The molecular signalling pathway was elucidated by analysing transcriptome. Additionally, the effects of increased Cornulin expression were also evaluated in an in vivo nude mice model. Results: The pre-treatment salivary Cornulin levels were significantly low (p=0.0001) in HNSCC patients (n=128,146.4±5.589pg/mL) and in oral premalignant disease patients (n=25, 174.6 ± 9.924 pg/mL) with respect to healthy controls (n=84,185.2±7.170pg/mL). Also, the tumor tissue expression of Cornulin (n=113,H-score=12.70±2.396) was significantly downregulated (p&amp;lt;0.0001) compared to the tumor-free margin (n=72,H-score=139.6±10.34). The patients showing complete clinical response regained normal salivary levels within 6 months of completion of treatment (p&amp;lt;0.0001). More importantly, low salivary Cornulin level at diagnosis was associated with poor overall survival (p=0.0282), indicating it to be a potential prognostic marker. On upregulation of Cornulin in low-expressing HNSCC cell lines (Cal27 and FaDu), the cellular viability, migration, and invasion showed a significant decrease. These indicate its potential as a tumor suppressor. RNAseq data of Cornulin overexpressed cell lines and further Gene set enrichment analysis, the G2/M checkpoint pathway was found to be positively enriched. Cornulin overexpression also arrests the G1/S phase of the cell cycle. GSEA and in vitro experiments revealed Cornulin to be involved in the PI3K/Akt/mTOR signalling pathway. In the nude mice xenograft model, decreased tumor progression, and tumor volume, was documented after Cornulin overexpression. There was an increase in differentiation in the xenograft tumors formed by Cornulin overexpressed cells. Conclusion: Here, we report for the first time that intrinsic Cornulin has a role as a potent antitumor and has potential as a prognostic marker. However the exact molecular mechanism needs to be further evaluated. Citation Format: Rajandeep Kaur, Anshika Chauhan, Sushmita Ghoshal, Jaimanti Bakshi, Radhika Srinivasan, Arnab Pal. Multifaceted role Cornulin in head and neck squamous cell carcinoma: From tumor suppressor pathophysiology to prognosticating biomarker [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 5595.

Abstract 3178: Vitamin B1 derivative, fursultiamine, prevents colon cancer growth

Abstract Colon cancer is the second deadliest type of cancer in the United States, with surgical resection being the primary treatment modality for localized early-stage colon cancer. Still, more treatment and prevention options are needed to help improve the outcomes for colon cancer as they are currently limited to invasive procedures. One promising option is fursultiamine, a disulfide lipid-soluble vitamin B1 derivative, which has anti-oxidative and anti-inflammatory activity that could aid in preventing colon cancer. We hypothesize that supplementation of fursultiamine could be an effective chemopreventive treatment to control the growth and spread of colon carcinogenesis. Human colorectal adenocarcinoma (SW480) cell lines were incubated with complete media (CM) or human recombinant endothelial growth factor (EGF 20 ng/ml) in the absence and presence of fursultiamine. The cell viability was examined by MTT assay in a concentration- (0-150 uM) and time (24 and 48 h) - dependent manner. Our results indicate that fursultiamine prevents SW480 cell death in a dose-dependent manner. Apoptosis was detected by using Annexin-V staining and live and death cell assay. Our results suggest that fursultiamine prevents SW480 cell proliferation by inducing apoptotic cell death. The production of reactive oxygen species (ROS) and activation of caspase-3 were examined by specific assay kits. Treatment with fursultiamine resulted in the decreased production of ROS and activation of caspase-3. The expression of anti-apoptotic, pro-apoptotic, and pro-inflammatory factors was analyzed using antibody arrays. The results demonstrated that treatment with fursultiamine increased the expression of pro-apoptotic factors such as Bad, Bax, cleaved caspase 3, Fas/CD95, and FADD. Further, fursultiamine increased expression of the tumor suppressor genes p53, p27, and p21. We next planned to examine this vitamin B1 derivative's effect in preventing cancer growth in nude mice xenograft models. In conclusion, our in vitro results indicate that fursultiamine, a vitamin B1 derivative, prevents colon cancer growth through increased regulation of pro-apoptotic pathways and tumor suppressor proteins and thus has the potential for further development as a chemopreventative agent. Citation Format: Sudeep Poludasu, Christian Pompoco, Kota Ramana. Vitamin B1 derivative, fursultiamine, prevents colon cancer growth [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 3178.

Abstract 431: Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer

Abstract Background &amp; Hypothesis: Treatments for triple-negative breast cancer (TNBC) are limited to chemotherapy, PARP inhibitors and immunotherapies. Tumor metabolism seeks to identify metabolic pathways preferentially utilized by cancer. Malic enzyme 2 (ME2) has a role in the malate-aspartate shuttle and pyruvate and NADPH production, contributing to the redox balance and metabolite synthesis. We demonstrated that ME2 knockdown inhibits growth of TNBC cells with upregulated serine biosynthesis pathway. We hypothesized that ME2 knock-down (kd) would be growth inhibitory in vivo. Methods: We used a non-tumorigenic control (MCF10a) and TNBC lines (BT20, MDA-MB-468, HCC1806, HCC70, Hs578T, SUM149). Nude mice received mammary fat pad xenografts of MDA-MB-468, to assess the impact of ME2-shRNA on growth. ME2 relevant metabolite profiling was achieved through the Promega Metabolite-Glo assay, measuring NAD, NADH, NADP, NADPH, Malate, and Glutamate. Cellular proliferation was quantified via the BioTek BioSpa 8 system and Promega CellTiter Glo. Comprehensive profiling of metabolic genes was achieved with the Nanostring nCounter across all cell lines. The potential therapeutic efficacy of NPD-389, MDSA, disodium embonate and doxorubicin was evaluated, alone or in combination, through targeted drug assays. Metabolic flux analyses, specifically mitochondrial and glycolytic function, were conducted using the Seahorse XF96, contrasting ME2-shRNA modified and control cells. Results: ME2 kd resulted in reduced proliferation in several TNBC cell lines to varying degrees, in contrast to control MCF10a, and to Hs578T. In response to the loss of ME2, several cell lines upregulated serine biosynthesis genes, including PSAT1. MDA-MB-468 ME2 kd cells developed significantly smaller tumors in a nude mouse xenograft model. Increased malate levels, but reduced NADH and NADPH, were detected in TNBC cells with inhibited growth post-ME2 kd, but not in MCF10a or Hs578T cells. Among the tested ME2 inhibitors, NPD-389 had superior efficacy vs. MDSA and disodium embonate. Metabolic flux analysis showed compromised mitochondrial and glycolytic processes in the TNBC cell lines exhibiting growth inhibition upon ME2 kd. Conclusions: ME2 knockdown selectively impairs cell proliferation and tumor growth in TNBC cell lines, validating its potential as a therapeutic target. The differential metabolic response, including altered malate and NADH/NADPH levels, confirms ME2's importance in TNBC metabolism. NPD-389 effectively reduced growth in ME2-sensitive lines, meriting further investigation. These findings support targeting ME2 in as a novel treatment strategy. Citation Format: Mark D. Slayton, Zackariah A. Farah, Jin Heon Jeon, Abhinav Achreja, Ben Krinkel, Brisilda Nilaj, Yi-Hsien Eu, Justin Sung, Alyssa Rosenfeld, Alisa Lui, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Kerry Loomes, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer [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 431.

Abstract 3585: DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine cancer

Abstract Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are neoplasms originating from the gastrointestinal track and pancreas. Unfortunately, most patients at the time of diagnosis have extensive metastatic disease and are not candidates for curative surgical resection of metastatic tumors. In such cases, targeted radiation therapy (RT) can slow the progression of metastatic disease, but eventually tumors develop resistance to radiation therapy. In this study, we examined whether DNA-PK inhibition could sensitize pancreatic NETs to radiation therapy and enhance the radiation therapy effect in a preclinical model of pNET metastasis. Methods. We used 96-well clonogenic assays to evaluate the efficacy of the DNA-PK inhibitor peposertib in combination with RT in vitro in QGP-1 and BON pancreatic NET cell lines. Double-strand break-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence after peposertib treatment alone or in combination with RT. Western blot analysis was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) combination RT. The efficacy of peposertib in combination with RT was evaluated in QGP-1 and BON human xenograft models in athymic nude mice and a BON preclinical lung metastasis model. Results. Clonogenic assays demonstrated absence of cytotoxicity from peposertib alone at doses below 1000 nM and a strong radiosensitizing effect at 200 nM in both QGP-1 and BON cell lines. Pretreatment with peposertib or treatment within 2 h after RT suppressed colony formation; treatment at later timepoints did not enhance radiotherapy. Immunofluorescence analysis revealed high numbers of γH2AX foci with peposertib therapy alone at 96 h or when given 2 h after RT. Western blot analysis confirmed inhibition of DNA-PK Ser2056 after RT in combination with peposertib. DNA-PK inhibition in combination with RT led to remarkable repression of pNET growth both in subcutaneous xenografts (&amp;gt;90%) and in the preclinical lung metastasis model (&amp;gt;90%). Conclusions. Our results showed several advantages of targeting DNA-PK to enhance radiation sensitivity in high-grade pNETs: (1) DNA-PK inhibition alone disrupts DNA damage response in pNETs as demonstrated by strong H2AX phosphorylation. (2) DNA-PK inhibition is most effective at enhancing radiotherapy either right before or after radiation therapy administration. This demonstrates that extended DNA-PK inhibition is not necessary to achieve marked responses during pNET radiotherapy. (3) DNA-PK inhibition 30 min before radiation therapy is sufficient to greatly reduce pNET tumor growth or metastasis progression in pNET preclinical models. In summary, selective DNA-PK inhibition provides a potent therapeutic strategy for disruption of non-homologous end joining DNA double-strand break repair and may offer a novel therapeutic approach in advanced NET radiotherapy. Citation Format: Piotr G. Rychahou, Aman Chauhan, Zeta Chow, Tadahide Izumi, Quan Chen, Eun Y. Lee, Dana Napier, Lowell Brian Anthony, Michael J. Cavnar, Courtney M. Townsend, B. Mark Evers. DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine cancer [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 3585.

Abstract 1938: WEE1 inhibition prevents and overcomes resistance to KRAS inhibitors in lung cancer by enhancing MCL1-mediated apoptosis

Abstract Background: While novel covalent inhibitors of KRAS G12C have been approved in non-small-cell lung cancer (NSCLC), their efficacies are known to be limited. To enhance the therapeutic potential of KRAS inhibitors, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination partners that induced synergistic efficacy with KRAS inhibitors. Additionally, mechanistic analyses were conducted to clarify the synergistic effects in combination and their role in conferring resistance to KRAS inhibitors. Methods: To identify novel combination partners of KRAS inhibitors, we conducted high-throughput screening (HTS) using a chemical library composed of 1,400 kinase inhibitors. Sotorasib was used as a KRAS inhibitor. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Protein expression levels were assessed by immunoblotting. In vivo studies were performed using a H358 xenograft nude mouse model and a patient-derived xenograft model (PDX). PDX samples were provided by the National Cancer Center J-PDX library, Japan. Results: HTS identified the WEE1 inhibitor AZD1775, a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of KRAS inhibitors in KRAS G12C mutant NSCLC. Synergistic effects of KRAS and WEE1 inhibitors were confirmed across multiple KRAS G12C mutant NSCLC cell lines, independently of their co-occurring oncogenic mutation profiles. The combination treatment enhanced apoptotic cell death, being upregulated pro-apoptotic protein BIM, while downregulated anti-apoptotic protein MCL1. In vivo efficacy studies in combination with sotorasib and AZD1775 also demonstrated remarkable tumor regression and durable response in both CDX and PDX models. We also generated sotorasib-resistant H23 (H23-SR) cells, confirming upregulation of both WEE1 and MCL1 in the resistant cells. Ectopic overexpression of WEE1 in H23, on the contrary, conferred resistance to sotorasib. Furthermore, WEE1 inhibition re-sensitized H23-SR cells to KRAS inhibitors. Conclusion: Our results suggest that the WEE1-MCL1 axis plays an important role in both the initial therapeutic efficacy and the acquired resistance to KRAS inhibitors. These findings could pave the way for a novel therapeutic strategy for KRAS G12C mutant NSCLC. Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Takehiro Nakao, Shunta Mori, Jie Liu, Susumu S. Kobayashi, Akihiro Ohashi. WEE1 inhibition prevents and overcomes resistance to KRAS inhibitors in lung cancer by enhancing MCL1-mediated apoptosis [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 1938.