Proliferation Of Cancer Cell Lines Research Articles

SOAT1 in gallbladder cancer: Clinicopathological significance and avasimibe therapeutics.

The aim of this investigation was to evaluate the differential expression of the sterol O-acyltransferase 1 (SOAT1) protein in gallbladder cancer tissues and cells, investigate the impact of Avastin on the proliferation, migration, invasion capabilities of gallbladder cancer cells, and its potential to induce cell apoptosis. Immunohistochemical analysis of samples from 145 gallbladder cancer patients was conducted, along with analysis of SOAT1 protein, mRNA expression levels, and cholesterol content in gallbladder cancer cell lines SGC-996, NOZ, and gallbladder cancer (GBC)-SD using Western blot and q-PCR techniques. Furthermore, the effects of Avastin on the proliferation, migration, and invasion capabilities of these gallbladder cancer cell lines were studied, and its ability to induce cell apoptosis was evaluated using flow cytometry, Western blot, and immunohistochemical methods. Additionally, gene expression and pathway analysis were performed, and the synergistic therapeutic effects of Avastin combined with gemcitabine were tested in a gallbladder cancer xenograft model. The study found that SOAT1 expression was significantly upregulated in GBC tissues and positively correlated with lymph node metastasis and TNM staging. In vitro experiments demonstrated that Avastin significantly inhibited the proliferation, migration, and invasion capabilities of SGC-996 and GBC-SD cell lines and induced apoptosis. RNA sequencing analysis revealed multiple differentially expressed genes in cells treated with Avastin, primarily enriched in biological pathways such as signaling transduction, malignant tumors, and the immune system. In vivo, experiments confirmed that Avastin could effectively suppress tumor growth in a gallbladder cancer xenograft model and enhanced the treatment efficacy when used in combination with gemcitabine. Overall, these findings provide new insights and strategies for targeted therapy in gallbladder cancer.

Pectinose induces cell cycle arrest in luminal A and triple-negative breast cancer cells by promoting autophagy through activation of the p38 MAPK signaling pathway

Breast cancer patients often have a poor prognosis largely due to lack of effective targeted therapy. It is now well established that monosaccharide enhances growth retardation and chemotherapy sensitivity in tumor cells. However, Pectinose whether has capability to restrict the proliferation of tumor cells remain unclear. Here, we report that Pectinose induced cytotoxicity is modulated by autophagy and p38 MAPK signaling pathway in breast cancer cell lines. The proliferation of cells was dramatically inhibited by Pectinose exposure in a dose-dependent manner, which was relevant to cell cycle arrest, as demonstrated by G2/M cell cycle restriction and ectopic expression of Cyclin A, Cyclin B, p21and p27. Mechanistically, we further identified that Pectinose is positively associated with autophagy and the activation of the p38 MAPK signaling in breast cancer. In contrast, 3-Ma or SB203580, the inhibitor of autophagy or p38 MAPK, reversed the efficacy of Pectinose suppressing on breast cancer cell lines proliferation and cell cycle process. Additionally, Pectinose in vivo treatment could significantly inhibit xenograft growth of breast cancer cells. Taken together, our findings were the first to reveal that Pectinose triggered cell cycle arrest by inducing autophagy through the activation of p38 MAPK signaling pathway in breast cancer cells,especially in luminal A and triple-negative breast cancer.

Transglutaminase 2 serves as a pathogenic hub gene of KRAS mutant colon cancer based on integrated analysis.

Colon cancer is acknowledged as one of the most common malignancies worldwide, ranking third in United States regarding incidence and mortality. Notably, approximately 40% of colon cancer cases harbor oncogenic KRAS mutations, resulting in the continuous activation of epidermal growth factor receptor signaling. To investigate the key pathogenic genes in KRAS mutant colon cancer holds considerable importance. Weighted gene co-expression network analysis, in combination with additional bioinformatics analysis, were conducted to screen the key factors driving the progression of KRAS mutant colon cancer. Meanwhile, various in vitro experiments were also conducted to explore the biological function of transglutaminase 2 (TGM2). Integrated analysis demonstrated that TGM2 acted as an independent prognostic factor for progression-free survival. Immunohistochemical analysis on tissue microarrays revealed that TGM2 was associated with an elevated probability of perineural invasion in patients with KRAS mutant colon cancer. Additionally, biological roles of the key gene TGM2 was also assessed, suggesting that the downregulation of TGM2 attenuated the proliferation, invasion, and migration of the KRAS mutant colon cancer cell line. This study underscores the potential significance of TGM2 in the progression of KRAS mutant colon cancer. This insight not only offers a theoretical foundation for therapeutic approaches but also highlights the need for additional clinical trials and fundamental research to support our preliminary findings.

Therapeutic potential of esculetin in various cancer types (Review).

Esculetin (Esc), a coumarin derivative and herbal medicinal compound used in traditional Chinese medicine, is extracted from Fraxinus chinensis. Esc has shown notable potential in the inhibition of proliferation, metastasis and cell cycle arrest in various cancer cell lines. The present review is based on research articles regarding Esc in the field of carcinoma, published between 2009 and 2023. These studies have unanimously demonstrated that Esc can effectively inhibit cancer cell proliferation through diverse mechanisms and modulate multiple signaling pathways, such as Wnt/β-catenin, PI3K/Akt, MAPK and janus kinase/signal transducer and activator of transcription-3. In addition, the safety profile of Esc has been demonstrated in credible animal experiments, which has indicated Esc as an effective compound. Furthermore, the combination therapy of Esc with commonly used chemotherapeutic drugs holds great promise. The aim of the present review was to encourage further studies and applications of Esc in cancer therapy.

Mechanistic Insights into Yifei Sanjie Pill's Regulation of EMT to Enhance Gefitinib Treatment effect in NSCLC by in silico analysis and experimental validation

Ethnopharmacological relevanceThe Yi-Fei San-Jie pill (YFSJ) is a well-known Chinese medicine that has been used to treat non-small cell lung cancer in China for decades. Aim of the studyPrevious studies have shown that YFSJ combined with gefitinib can effectively inhibit the proliferation of gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines by promoting apoptosis and autophagy, but the molecular biological mechanisms involved and whether YFSJ combined with gefitinib can have synergistic effects still need to be further explored. Thus, the present study aimed to establish an in silico and experimental framework to decipher the underlying mechanism by which YFSJ augments the efficacy of gefitinib in treating NSCLC. Materials and methodsIntegrated approaches, including microarray analysis, network pharmacology, RNA sequencing, bioinformatics algorithm analysis and in vivo and in vitro experiments, were applied to elucidate the underlying mechanism. ResultsAnalysis of microarray datasets indicated that gefitinib may play a role in the regulation of the epithelial–mesenchymal transition (EMT) of PC9 cells. EMT-related Gene Ontology (GO) terms and the MAPK pathway were found to be enriched in the differentially expressed genes (DEGs), and a decreasing trend was observed in the EMT score. Network pharmacology analysis revealed that the potential NSCLC-related targets of YFSJ also showed enrichment in EMT-related GO terms and the MAPK pathway. Experimental findings demonstrated that combined YFSJ-treated serum and gefitinib treatment significantly inhibited PC9 cell migration and invasion. In addition, the combined treatment dramatically reduced the tumour volume in an animal model. The effectiveness of the combination treatment surpassed that of gefitinib alone in both cell and animal experiments. RNA sequencing analysis revealed significant enrichment of DEGs in EMT-related GO terms for the gefitinib treatment group, YFSJ treatment group, and combination treatment group compared to the control group. Notably, the negative regulation of EMT showed significant enrichment in the DEGs of the combination treatment group. The MAPK pathway was significantly enriched among the different groups. Moreover, combined treatment with YFSJ and gefitinib may exert synergistic anti-NSCLC effects by inhibiting the p-p38 MAPK/GSK3β signalling axis, subsequently suppressing downstream EMT processes. ConclusionCombined treatment with YFSJ and gefitinib could enhance the sensitivity of NSCLC cells to gefitinib by suppressing EMT through the EGFR/p-p38 MAPK/GSK3β signalling axis. YFSJ may serve as an important adjunctive medication for NSCLC patients receiving gefitinib treatment in clinical practice

RAS isoform specific activities are disrupted by disease associated mutations during cell differentiation

The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation.Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.

Evaluating the Anti-proliferative and Apoptotic Role of Atrial Natriuretic Peptide in Colon Cancer Cell Lines

The use of small peptides and conventional anticancer drugs is gaining importance in oncology as small peptides may help increase chemo or radiation sensitivity. The present study aimed to study the impact of atrial natriuretic peptide (ANP) in reducing colon cancer cell proliferation in primary and metastatic colon cancer cell lines. The proliferation of colon cancer cell lines (SW480 and SW620) was analysed by CCK-8 assay and cell damage was analyzed by lactate dehydrogenase activity. Catalase activity assay was performed to measure the oxidative stress and antioxidant defense mechanisms in SW480 and SW620 cell lines. Subsequently, up or downregulation of cancer-specific gene expression such as BAX, Caspase-3, BCL-2, CDK-6, and PCNA genes were assessed after the treatment of small peptide ANP in SW480 and SW620 colon cancer cell lines. The ANP treatment decreased the colon cancer cell proliferation, upregulated the apoptosis-related gene expression (Caspase-3, BAX), downregulated the anti-apoptotic gene (BCL-2) expression, and proliferation-related genes (CDK-6, PCNA) in SW480 and SW620 colon cancer cell lines, and the differences were found to be statistically significantly. Further, increased levels of catalase in the colon cancer cell lines after ANP-treatment suggested the therapeutic role of ANP. Subsequently, the LDH analysis showed the potential of ANP in inducing colon cancer cell damage. Collectively, the current study clearly shows that ANP is a potential molecule in reducing uncontrolled cancer cell growth. However, additional research using animal models and additional colon cancer cell lines is needed to validate its potential usage in clinical studies.

Ethaverine and Papaverine Target Cyclin-Dependent Kinase 5 and Inhibit Lung Cancer Cell Proliferation and Migration.

CDK5 kinase plays a central role in the regulation of neuronal functions, and its hyperactivation has been associated with neurodegenerative pathologies and more recently with several human cancers, in particular lung cancer. However, ATP-competitive inhibitors targeting CDK5 are poorly selective and suffer limitations, calling for new classes of inhibitors. In a screen for allosteric modulators of CDK5, we identified ethaverine and closely related derivative papaverine and showed that they inhibit cell proliferation and migration of non small cell lung cancer cell lines. Moreover the efficacy of these compounds is significantly enhanced when combined with the ATP-competitive inhibitor roscovitine, suggesting an additive dual mechanism of inhibition targeting CDK5. These compounds do not affect CDK5 stability, but thermodenaturation studies performed with A549 cell extracts infer that they interact with CDK5 in cellulo. Furthermore, the inhibitory potentials of ethaverine and papaverine are reduced in A549 cells treated with siRNA directed against CDK5. Taken together, our results provide unexpected and novel evidence that ethaverine and papaverine constitute promising leads that can be repurposed for targeting CDK5 in lung cancer.

Differential Effect of 4H-Benzo[d] [1, 3]oxazines on the Proliferation of Breast Cancer Cell Lines.

A family of 4H-benzo[d][1,3]oxazines were obtained from a group of N-(2-alkynyl)aryl benzamides precursors via gold(I) catalysed chemoselective 6-exo-dig C-O cyclization. The precursors and oxazines obtained were studied in breast cancer cell lines MCF-7, CAMA-1, HCC1954 and SKBR-3 with differential biological activity showing various degrees of inhibition with a notable effect for those that had an aryl substituted at C-2 of the molecules. 4H-benzo[d][1,3]oxazines showed an IC50 rating from 0.30 to 157.4 µM in MCF-7, 0.16 to 139 in CAMA-1, 0.09 to 93.08 in SKBR-3, and 0.51 to 157.2 in HCC1954 cells. We observed that etoposide is similar to benzoxazines while taxol effect is more potent. Four cell lines responded to benzoxazines while SKBR-3 cell line responded to precursors and benzoxazines. Compounds 16, 24, 25 and 26 have the potent effect in cell proliferation inhibition in the 4 cell lines tested and correlated with oxidant activity suggesting a possible mechanism by ROS generation. These compounds represent possible drug candidates for the treatment of breast cancer. However, further trials are needed to elucidate its full effect on cellular and molecular features of cancer.

Phytosynthesis of silver nanoparticles using aqueous sandalwood (Santalum album L.) leaf extract: Divergent effects of SW-AgNPs on proliferating plant and cancer cells.

The biogenic approach for the synthesis of metal nanoparticles provides an efficient eco-friendly alternative to chemical synthesis. This study presents a novel route for the biosynthesis of silver nanoparticles using aqueous sandalwood (SW) leaf extract as a source of reducing and capping agents under mild, room temperature synthesis conditions. The bioreduction of Ag+ to Ago nanoparticles (SW-AgNPs) was accompanied by the appearance of brown color, with surface plasmon resonance peak at 340-360 nm. SEM, TEM and AFM imaging confirm SW-AgNP's spherical shape with size range of 10-32 nm. DLS indicates a hydrodynamic size of 49.53 nm with predominant negative Zeta potential, which can contribute to the stability of the nanoparticles. FTIR analysis indicates involvement of sandalwood leaf derived polyphenols, proteins and lipids in the reduction and capping of SW-AgNPs. XRD determines the face-centered-cubic crystalline structure of SW-AgNPs, which is a key factor affecting biological functions of nanoparticles. This study is novel in using cell culture methodologies to evaluate effects of SW-AgNPs on proliferating cells originating from plants and human cancer. Exposure of groundnut calli cells to SW-AgNPs, resulted in enhanced proliferation leading to over 70% higher calli biomass over control, enhanced defense enzyme activities, and secretion of metabolites implicated in biotic stress resistance (Crotonyl isothiocyanate, Butyrolactone, 2-Hydroxy-gamma-butyrolactone, Maltol) and plant cell proliferation (dl-Threitol). MTT and NRU were performed to determine the cytotoxicity of nanoparticles on human cervical cancer cells. SW-AgNPs specifically inhibited cervical cell lines SiHa (IC50-2.65 ppm) and CaSki (IC50-9.49 ppm), indicating potential use in cancer treatment. The opposing effect of SW-AgNPs on cell proliferation of plant calli (enhanced cell proliferation) and human cancer cell lines (inhibition) are both beneficial and point to potential safe application of SW-AgNPs in plant cell culture, agriculture and in cancer treatment.

Protocol for Testing the Effects of ssDNA Aptamer in HeLa and MCF-7.

Conventional approaches for treating tumors encompass chemotherapy, radiotherapy, and surgery. However, these methods come with their limitations when applied in clinical practice. Aptamers are often referred to as "chemical antibodies" and consist of short DNA or RNA molecules, designed to bind to a wide range of targets, including proteins or nucleic acid structures. They exhibit strong affinities and remarkable specificity for their target molecules, making them capable of functioning as therapeutic agents to directly impede tumor cell proliferation. This approach helps minimize the harm to normal cells, thus reducing toxicity through decreased side effects. Here we report the procedure to develop ssDNA aptamer and investigate its ability to inhibit cancer cell proliferation in HeLa and MCF-7 cancer cell lines.

Abstract LB158: Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy

Abstract Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines and comparison of the effects of each drug combination with the effect of the individual drugs are shown in Table. The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes. Table. Colony formation and drug-mediated inhibition of cell proliferation in pancreatic cancer cell lines. Comparison of the effects of each drug combination with the effect of the individual drugs (Table shows the p-values for each comparison.) Colony formation BxPC-3 PL45 Capan-1 Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano SAHA TLZ Ola DAC Pano+TLZ 0.00048 0.0052 2.40E-06 2.70E-06 0.0051 0.021 Pano+Ola 0.0035 0.078 0.0011 0.00094 0.0016 0.022 SAHA+TLZ 1.80E-05 0.17 0.0029 0.012 0.0014 0.019 SAHA+Ola 0.00018 0.014 0.0016 0.0068 0.0066 0.029 Pano+TLZ+DAC 4.20E-05 0.00026 0.0015 1.70E-05 1.20E-05 2.70E-06 0.00052 0.00088 0.0064 Pano+Ola+DAC 3.40E-05 0.00049 0.0013 7.10E-07 1.70E-06 2.20E-06 0.00068 0.0028 0.0053 SAHA+TLZ+DAC 0.00016 0.00016 0.0011 1.90E-05 3.70E-06 9.30E-07 0.00018 0.00017 0.011 SAHA+Ola+DAC 4.90E-07 0.00035 0.001 0.00022 0.00026 0.00084 0.00016 0.0016 0.0035 Drug-mediated inhibition of cell proliferation Pano+TLZ+DAC 3.57E-05 3.89E-07 1.30E-07 2.91E-05 3.76E-06 1.80E-08 0.0239 ND 0.0092 Pano+Ola+DAC 7.76E-05 1.43E-07 2.52E-07 6.59E-07 1.03E-08 2.22E-07 0.0013 0.21 0.0034 SAHA+TLZ+DAC 3.04E-06 0.00034 1.17E-05 4.91E-08 4.45E-06 1.74E-07 0.0013 1.75E-01 0.007 SAHA+Ola+DAC 0.00012 0.0032 0.000068 9.64E-09 1.53E-08 9.35E-07 0.0007 0.18 0.0054 Abbreviations: DAC: decitabine; ND: not determined; Ola: olaparib; Pano: panobinostat; SAHA: vorinostat; TLZ: talazoparib P ≤ 0.05 is considered statistically significant. Citation Format: Apostolia M. Tsimberidou, Benigno C. Valdez, Bin Yuan, Yago Nieto, Mehmet Baysal, Abhijit Chakraborty, Borje S. Andersson. Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: implications for novel therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB158.

Abstract ND02: Discovery of BMS-986365, a ligand-directed androgen receptor degrader (AR LDD) with a dual mechanism-of-action and best-in-class potential, for the treatment of advanced prostate cancer

Abstract The androgen receptor (AR) is a key driver of the cellular processes that contribute to the pathology of prostate cancers at most stages, and drugs that inhibit the activity of this receptor or interfere with androgen biosynthesis have become the cornerstone of treatments for prostate cancer. While both types of therapy have had a significant positive impact on disease progression and overall survival, de novo and acquired resistance remains an impediment to a durable clinical response in patients with metastatic castration-resistant prostate cancer (mCRPC). The mechanisms underlying drug resistance are complex and multifactorial, being attributable to AR overexpression, point mutations in the receptor that alter drug pharmacology, and to the upregulation of receptor-associated coregulators, which render AR less dependent on an activating ligand. Regardless, absolute inhibition of AR signaling remains the objective of contemporary approaches to treat prostate cancer. Recently, there has been specific interest in the development of approaches to eliminate AR proteins using drugs that enable targeted degradation of AR in cancer cells.This puts in context the significance of our discovery of BMS-986365 (also known as CC-94676), a heterobifunctional ligand-directed degrader (LDD) that enables the CRL4CRBN E3 ligase-dependent ubiquitination and degradation of AR (DC50 10 to 40 nM, Ymin 7 to 19%). BMS-986365 is a highly potent and selective AR degrader that induces rapid and deep degradation of both wildtype and mutant forms of the receptor residing in either the cytoplasmic or nuclear compartments of the cell. The drug is ~100-fold more potent than enzalutamide (ENZ) at inhibiting androgen-stimulated transcription of AR target genes, and 10 to 120-fold more potent than ENZ at inhibiting AR-dependent proliferation of multiple prostate cancer cell lines in vitro. In animal models of advanced prostate cancer, BMS-986365 demonstrates on-target activity, degrading AR, suppressing AR signaling, and inhibiting tumor growth. Indeed, tumor volume reductions of 63-92% were achieved by BMS-986365 in validated models of advanced CRPC and therapy resistant patient-derived xenografts, including those with acquired resistance to ENZ. Collectively our preclinical data suggest that the AR degrader BMS-986365 is superior to standard-of-care AR antagonists, such as ENZ, in both preclinical and disease-relevant animal models, and support its clinical development for treatment of prostate cancer. BMS-986365 has advanced into clinical studies where it has demonstrated encouraging clinical activity in patients with mCRPC. Citation Format: Shuichan Xu, Surendra Nayak, John D. Norris, Massimo Ammirante, Emyly Rychak, Suzanne E. Wardell, Toshiya Tsuji, Ken Liu, Joseph Meiring, Joseph R. Piccotti, Deepak Dalvie, Debbie Liao, Raju Kandimalla, Nadia Guerrero, Lisa Sapinoso, Jennifer G. Baker, Yeeun Bae, Joshua Baughman, Brandon Toyama, Celia Fontanillo Fontanillo, Stephen Norris, Evan J. Horn, Veronique Plantevin-Krenitsky, Deborah Mortensen, Brian Cathers, Marie Hong Nguyen, Joshua D. Hensen, Lawrence G. Hamann, Donald P. McDonnell, Rama Krishna Narla, Mark Rolfe. Discovery of BMS-986365, a ligand-directed androgen receptor degrader (AR LDD) with a dual mechanism-of-action and best-in-class potential, for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND02.

Abstract LB177: Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC

Abstract Prostate cancer is the second most commonly diagnosed cancer and the second leading cause of cancer death among men worldwide. Androgens play an important role in the development and progression of prostate cancers and targeting androgen receptor (AR) signaling axis over decades has remained a mainstay of prostate cancer therapy. Next generation hormonal agents (NHAs) enzalutamide and abiraterone have brought clinical benefits for metastatic castrate-resistant prostate cancer (mCRPC) patients, AR axis-targeted therapy resistance is inevitable due to AR gene amplification or mutations. AR-V7 is the most clinically relevant AR splice variant associated with endocrine resistance and poor prognosis. AR-V7 constitutively activates AR signaling and lacks the ligand binding domain (LBD) where hormones and AR antagonists interact, resulting in resistance to AR signaling inhibitors (ARSi). Thus, AR-V7 is a promising therapeutic target to address ARSi resistance in mCRPC. We have discovered a highly potent AR-V7 proteolysis targeting chimera (PROTAC), HC-4955 that degrades both the wild-type AR (WT-AR) and AR-V7 mutant. HC-4955 degrades AR-V7 with DC50 < 5 nM. In silico molecular docking analysis predicts the binding of HC-4955 to the N-terminal domain of AR and then the essential amino acids contributed to HC-4955 binding are validated by mutational analysis studies. HC-4955 also potently degrades mutants of AR-LBD mutations including L702H and T878A and these mutations are also associated with ARSi resistance. HC-4955 strongly inhibits proliferation of ARSi-resistant prostate cancer cell lines with IC50 < 10 nM. HC-4955 shows a robust anti-tumor activity in the AR-V7-bearing 22Rv1 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models, with majority of the tumors completely regressed and > 90% AR-V7 expression decreased at 1-5 mg/kg (QD, PO) in these models. The pre-clinical results support the clinical development of HC-4955 for the treatment of mCRPC. Selected pre-clinical data along with the chemical structure of HC-4955 will be presented. Citation Format: Jing Li, Zhilin Tu, Wu Du, Xi Xiang, Zeyu Wen, Yang You, Lijuan Zhao, Yin Chen, Chengcheng Yang, Hongwei Yuan, Xinghai Li. Discovery of HC-4955, a novel AR-V7-targeting PROTAC for the treatment of mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB177.

Abstract LB259: Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer

Abstract Epigenetic regulation plays an important role in prostate cancer progression and metastasis. KDM5D is a male-specific histone-modifying enzyme encoded on the Y chromosome that demethylates di- and tri-methylated forms of lysine 4 in histone H3, resulting in transcriptional repression of certain genes. In this study, we investigated the role of KDM5D in resistance to inhibitors targeting the G2/M cell cycle checkpoint kinase CHK1. Loss of KDM5D expression was associated with reduced cell proliferation and resistance to the CHK1 inhibitor SRA737 in castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer cell lines. In KDM5D expressing cells, depletion of KDM5D differentially impacted the proliferation of prostate cancer cell lines. The androgen-sensitive LNCaP cell line was particularly sensitive to the knockdown of KMD5D. On the other hand, in CRPC cell line 22Rv1, the knockdown of KDM5D caused resistance to both inhibitors, indicating a dual role of KDM5D in mediating ATR and CHK1 inhibitor sensitivity and cell proliferation in prostate cancer cells. Further analysis demonstrated a self-regulatory cycle between CHK1 and KDM5D in 22Rv1 cells, where depletion of KMD5D reduced CHK1 mRNA, while decreased CHK1 caused upregulation of KDM5D and overexpression of KDM5D resulted in increased CHK1 expression. The relevance of this regulatory mechanism to ATR and CHK1 inhibitor sensitivity is currently under investigation, along with several potential mediators of the differential responses to these inhibitors. Additional ATR and CHK1 inhibitors will also be examined in this context. This study may uncover novel biomarkers for ATR and CHK1 inhibitors that are currently in clinical trials for various cancers. KDM5D may be used to stratify prostate cancer patients receiving these highly potent anticancer agents. Citation Format: Wenxiao Zheng, Yuzhou Chen, Qiming J. Wang. Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB259.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Estrogen-receptor status determines differential regulation of α1- and α2-adrenoceptor-mediated cell survival, angiogenesis, and intracellular signaling responses in breast cancer cell lines.

Psychosocial stress promotes cancer pathogenesis involving angiogenesis through alterations in neuroendocrine-immune functions that may involve adrenoceptor (AR)-dependent signaling mechanisms in the brain, lymphoid organs, and cancerous cells. Various concentrations of α1- and α2- AR-specific agonists and antagonists were incubated in vitro with estrogen receptor-positive (ER +) MCF-7, and ER (-) MDA MB-231 cells to examine the secretions of VEGF-A, VEGF-C, and nitric oxide (NO), and expression of signaling molecules- p-ERK, p-CREB, and p-Akt on the proliferation of breast cancer cell lines. Cellular proliferation, VEGF-A and NO secretion, expression of p-ERK, p-CREB, and p-Akt were enhanced in MCF-7 cells treated with α1-AR agonist while VEGF-C secretion alone was enhanced in MDA MB-231 cells. Treatment of MCF-7 and MDA MB-231 cells with α2- AR agonist similarly enhanced proliferation and decreased NO production and p-CREB expression while VEGF-C secretion was decreased in MCF-7 cells and p-Akt expression was decreased in MDA MB-231 cells. α1-AR inhibition reversed cellular proliferation and VEGF-A secretion by MCF-7 cells while α2-AR inhibition reversed the proliferation of MCF-7 and MDA MB-231 cells and VEGF-C secretion by MCF-7 cells. Taken together, breast cancer pathogenesis may be influenced by distinct α-AR-mediated signaling mechanisms on angiogenesis and lymphangiogenesis that are dependent on estrogen receptor status.

Abstract 3195: SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in xenograft models

Abstract Background: Current clinical HER2-targeting ADCs (i.e. DS-8201) have shown promising clinical efficacy in HER2 expressing or mutant cancer patients. However, most of them will ultimately experience disease progression due to different mechanisms. Herein, we presented a novel HER2 ADC, SHR-4602, which demonstrated good in vitro and in vivo anti-tumor activities and could overcome the resistance caused by HER2 ADC including DS-8201 analogue (synthesized using DS-8201 structure) and SHR-A1811 (composed of trastuzumab, a cleavable linker, and a Topo I inhibitor payload, in phase III clinical trials) in xenograft models. Method: We used a stable enzyme-cleavable linker technology to conjugate the HER2 ADC using the amino acid sequence of pertuzumab and ER300 (eribulin derivative). The in vitro biological and anti-tumor activities were assessed by a series of assays. The in vivo anti-tumor efficacies were evaluated in the JIMT-1 (HER2 low expressing) as well as the SHR-A1811 and DS-8201 analogue induced JIMT-1 resistance (caused by treating with SHR-A1811 followed by DS-8201 analogue for a long-term) xenograft models in BALB/c nude mice. Results: Through the SPR assay, SHR-4602 exerted single nanomolar binding affinity to human and rhesus monkey HER2, but no binding activity against rat HER2. It demonstrated varying inhibitory activity on the proliferation of cancer cell lines with various HER2 expression levels, with the IC50 at the range of 0.007 nM to 8.21 nM. SHR-4602 showed highly efficient internalization in NCI-N87 HER2 expressing cells and could be translocated to the lysosome followed by releasing ER300. The cytotoxic activity of SHR-4602 was predominantly mediated by released ER300 through microtubule dynamics inhibition, G2/M arrest and apoptosis induction. Furthermore, SHR-4602 showed a strong in vitro bystander effect in the co-culture of HER2 positive/negative cells. SHR-4602 showed significant anti-tumor efficacy in JIMT-1, SHR-A1811 and DS-8201 analogue both-resistant JIMT-1 xenograft models. In SHR-A1811 and DS-8201 analogue both-resistant xenograft models, treatment with 10 mg/kg SHR-A1811 or DS-8201 analogue exerted negligible anti-tumor activity (TGI% < 17%), whereas SHR-4602 at 2.5, 5 and 10 mg/kg significantly inhibited the tumor growth (p < 0.001, vs. vehicle), with TGI% of 84.96%, 101.64% and 113.11% on Day 42, respectively, which indicated a superior anti-tumor efficacy against SHR-A1811 and DS-8201 analogue both-resistant xenograft models. Conclusion: SHR-4602 represents a new generation HER2-targeting ADC to deliver a potent cytotoxin to HER2 expressing tumor cells and may be more efficacious to the tumors in which current anti-HER2 therapies are ineffective. SHR-4602 is now in the phase I clinical development stage for advanced malignant solid tumors (NCT05819684). Citation Format: Yunan Tian, Xing Sun, Changyong Yang, Cheng Liao. SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in xenograft models [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 3195.

Abstract 7298: A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels

Abstract Introduction: Extra virgin olive oil is a basic component of the Mediterranean diet; it contains several molecules with antioxidant, anti-inflammatory, anti-angiogenic and pro-apoptotic properties against a variety of cancers and influencing cellular metabolism too. Olive mill waste water (OMWW), a liquid residue produced in vast amounts during olive oil extraction, represents an environmental issue but on the other side it is a rich source of phytochemicals with potential health benefits. Particularly, OMWWs are rich in different phenolic compounds, that show promise in preventing the development of several disease disorders, including cancer. We have assessed the anti-angiogenic and anti-tumor properties exerted by a nutraceutical from purified OMWW extract, in in vitro and in vivo models, and evaluated its effect in a human pilot study upon informed consent. Experimental procedures: OMWWs ability to affect cell proliferation and survival on different cancer cell lines was assessed by MTT. OMWWs interference with human umbilical endothelial vein cells (HUVECs) tube formation, migration and invasive capacities was studied by endothelial cell morphogenesis and migration assays. The inhibition of angiogenesis and tumor cell growth was investigated by the matrigel assay and tumour xenograft. To evaluate contribution of potential metabolomic biomarkers, SANIST (surface-activated chemical ionization-electrospray-NIST technology) mass spectrometric data-based platform has been employed on sera from a pilot study (Aiello et al, in preparation). Results: OMWW extract shown a potent inhibitory effect inhibiting cells proliferation of both cancer cell lines and HUVECs.OMWW inhibited several molecular targets expression, including IL8, Angiogenin, mTOR, VEGF, CXCR4, CXCL12 and CXCL8, in tumor cells and in TNF-α-stimulated HUVEC. We nalyzed sera from volunteers, after nutraceutical intervention with Oliphenoliaand we assessed several biological parameters. Endogenous vitamin D was significantly increased, as well as albumin, direct bilirubin, and calcium. We also observed glycemia and potassium decrease after Oliphenolia assumption. A previously unknow form of Vitamin D was also detected by mass spectrometry. Conclusions: OMWW extract has shown a stronger anti-angiogenic and anti-tumor properties, with potential chemo-preventive potentials. Results of human pilot study shows Oliphenolia as a promising and interesting natural dietary supplement, rich in polyphenol compounds, that can exert anti-inflammatory and antioxidant activities. Acknowledgements: This study was supported by a donation from the Fattoria La Vialla di Gianni, Antonio e Bandino Lo Franco - SAS (Arezzo, Italy). We are grateful to Professor Anna Aiello, and Professor Calogero Caruso, Palermo University for the pilot study. Citation Format: Adriana Albini, Luana Calabrone, Francesca Albini, Paola Corradino, Simone Cristoni, Douglas M. Noonan. A nutraceutical supplement derived from olive oil byproducts exhibits potential cancer preventive properties in preclinical studies and increases vitamin D levels [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 7298.

Abstract 1819: Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer

Abstract Background: Esculetin is a naturally occurring dihydroxycoumarin with anti-inflammatory, antioxidant, and anti-tumor activities. Copper ion transport proteins, notably SLC31A1 and ATP7B, are essential in maintaining intracellular copper homeostasis, averting disruptions that might lead to copper-induced cell death under normal physiological conditions. However, prior research has not explored Esculetin's role in inducing copper-induced cell death. Hence, this study aims to investigate the biological process of Esculetin-induced cell death and the molecular mechanism underlying dysregulation of intracellular copper ion homeostasis. Methods: Initially, the CCK-8 assay was used to determine the IC50 values of Esculetin on colorectal cancer cell lines (HCT116, HT29, SW480) and normal colonic epithelial cell lines (NCM460). In terms of biological behaviors, the effects of Esculetin on the proliferation of colorectal cancer cell lines were assessed using CCK-8, colony formation assay, and EdU staining. Flow cytometry was used to examine the impact of Esculetin on apoptosis and cell cycle distribution in colorectal cancer cells. A subcutaneous xenograft tumor model in nude mice was employed to evaluate the in vivo anti-tumor effects of Esculetin, while IHC staining was conducted to detect Ki-67 levels, and Western blotting was performed to assess EMT-related protein expression. To investigate copper ion homeostasis, qPCR was used to measure the expression levels of copper ion transporters SLC31A1 and ATP7B before and after Esculetin treatment. Results: The CCK-8 results revealed that the IC50 of Esculetin for normal colonic epithelial cell lines was significantly higher than that for colorectal cancer cell lines. Meanwhile, a notable inhibitory effect of Esculetin on the proliferation of colorectal cancer cells was observed. Additionally, Esculetin treatment suppressed colony formation and reduced the proportion of EdU-positive cells in colorectal cancer cells. Flow cytometry demonstrated an increase in apoptosis and S-phase arrest following Esculetin treatment. The subcutaneous xenograft tumor model showed significant inhibition of tumor growth by Esculetin, as evidenced by reduced Ki-67-positive cells in the Esculetin-treated group. Western blot analysis revealed a significant decrease in the expression levels of Vimentin and N-Cadherin and an increase in E-Cadherin expression. Furthermore, qPCR analysis post-Esculetin treatment showed an elevation in the inward copper transporter SLC31A1 levels, while the outward transporter ATP7B expression decreased. Conclusion: Esculetin induces dysregulation of intracellular copper ion homeostasis by upregulating SLC31A1 expression and downregulating ATP7B expression, leading to intracellular copper accumulation and subsequent disruption of copper homeostasis, thereby inducing copper-induced cell death. Citation Format: Fangyue Guo, Mengying Liu, Kaihe Zhu, Jiapeng Ru, Pei Li, Shanfeng Zhang. Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal 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 1819.