TNBC Cell Lines Research Articles

Identification of the metabolic protein ATP5MF as a potential therapeutic target of TNBC

BackgroundTriple-negative breast cancer (TNBC), a distinct subtype of breast cancer, is characterized by its high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Effective treatment regimens for non-BRCA1/2 mutation TNBC are still lacking. As a result, there is a pressing clinical necessity to develop novel treatment approaches for non-BRCA1/2 mutation TNBC.MethodsFor this research, the scRNA data was obtained from the GEO database, while the transcriptome data was obtained from the TCGA and METABRIC databases. Quality control procedures were conducted on single-cell sequencing data. and then annotation and the Copycat algorithm were applied for anlysis. Employing the high dimensional weighted gene coexpression network analysis (hdWGCNA) method, we analyzed the tumor epithelial cells from non-BRCA1/2 mutation TNBC to identify the functional module genes. PPI analysis and survival analysis were further emplyed to identify the key gene. siRNA-NC and siRNA-ATP5MF were transfected into two MDA-MB-231 and BT-549 TNBC cell lines. Cell growth was determined by CCK8 assay, colony formation and migration assay. Electron microscopy was used to examine the structure of mitochondria in cells. JC-1 staining was used to measure the potential of the mitochondrial membrane. A tumor xenograft animal model was established by injecting TNBC cells into nude mice. The animal model was usded to evaluated in vivo tumor response aftering ATP5MF silencing.ResultsUsing hdWGCNA, we have identified 136 genes in module 3. After PPI and survival analysis, we have identified ATP5MF as a potential therapeutic gene. High ATP5MF expression was associated with poor prognosis of non-BRCA1/2 mutation TNBC. The high expression of ATP5MF in TNBC tissues was evaluated using the TCGA database and IHC staining of clinical TNBC specimens. Silencing ATP5MF in two TNBC cell lines reduced the growth and colony formation of TNBC cells in vitro, and hindered the growth of TNBC xenografts in vivo. Additionally, ATP5MF knockdown impaired mitochondrial functions in TNBC cells.ConclusionIn summary, the metabolic protein ATP5MF plays a crucial role in the non-BRCA1/2 mutation TNBC cells, making it a potential novel diagnostic and therapeutic oncotarget for non-BRCA1/2 mutation TNBC.

MiR-17–5p/STAT3/H19: A novel regulatory axis tuning ULBP2 expression in young breast cancer patients

Background and aimUL-16 binding protein 2 (ULBP2) is a highly altered ligand for the activating receptor, NKG2D in breast cancer (BC). However, the mechanism behind its de-regulation in BC patients remains to be explored. The sophisticated crosstalk between miR-17–5p, the lncRNA H19, and STAT3 as a possible upstream regulatory loop for ULBP2 in young BC patients and cell lines remains as an unexplored area. Therefore, this study aimed at unravelling the ncRNA circuit regulating ULBP2 in young BC patients and cell lines. Patients and methodsA total of 30 BC patients were recruited for this study. The expression levels of miR-17–5p, lncRNA H19, and STAT3 were examined in 30 BC tissues compared to their normal counterparts. In addition, the expression signatures of those transcripts were compared in young (<40 years) and old BC (≥40 years) patients. miR-17–5p oligonucleotides, STAT3 and H19 siRNAs were transfected in MDA-MB-231 cells using HiPerfect® Transfection Reagent. miR-17–5p and the transcripts of the target genes quantified using RT-qPCR. Their relative expression was calculated using the 2–ΔΔCT method. ResultsThrough acting as a ceRNA circuit that antagonizes the function of miR-17–5p, H19 prevented the miR-17–5p-induced downregulation of STAT3; this mechanism further contributes to the pathogenesis of BC. Ectopic expression of miR-17–5p in MDA-MB-231 cells displayed its prominent role as an indirect potential activator of NK cells by significantly repressing the expression levels of the oncogenic mediator STAT3 and the oncogenic lncRNA H19 and inducing ULBP2 expression level by 3 folds in TNBC cell lines compared to mock cells. Furthermore, knocking down of STAT3 repressed the lncRNA H19 and increased ULBP2 expression levels, whereas siRNAs against H19 increased the expression levels of ULBP2. ConclusionThis study highlighted the crosstalk between the novel regulatory network composed of miR-17–5p, H19 and STAT3, and their impact on ULBP2 in BC. Moreover, this study underscored the potential role of miR-17–5p in counteracting the immune evasion tactics, particularly the shedding of ULBP2 in young BC patients, through the modulation of the STAT3/H19/ULBP2 regulatory axis. Thus, targeting this novel regulatory network could potentially enhance our understanding and advance the future application of the innate system-mediated immunotherapy in BC.

Knockout of histone deacetylase 8 gene in breast cancer cells may alter the expression pattern of the signaling molecules

Breast cancer (BC) is the most common cancer diagnosed in the world and it is also the main leading cause of cancer deaths in women. Change in epigenetic mechanisms promotes BC initiation and progression. Histone deacetylase 8 (HDAC8) was found to act as a potential oncogene in different malignancies. For better understanding of the HDAC8 function in BC development, we investigated the effect of HDAC8 deletion on the expression of genes involved in signaling pathways. In this study, CRISPR technology was used to knockout the HDAC8 gene in MDA-MB-468, MDA-MB-231 and MCF-7 cell lines. For this purpose, two gRNAs were designed and cloned into the PX459 vector. The gRNA-containing vectors were transfected into the BC cell lines and then the effect of this deletion on the expression of genes involved in signaling pathway was determined using quantitative real-time PCR (qRT-PCR). Analysis of qRT-PCR results showed a reduction in the expression of studied genes in BC cell lines after deletion of the HDAC8 gene compared to untreated controls. Although this decline was not significant for FGF2 and FGFR1 genes, however the mTOR, IGF1R, INSR, VEGFA and VEGFR2 genes showed statistically significant reduction in the studied BC cell lines. In addition, the down-regulation of PDGFC and PDGFRA genes were only significant in the TNBC cell lines. Overall, our study showed that HDAC8 can exert its oncogenic effects by altering the expression level of molecules involved in some signaling pathways, and inhibiting HDAC8 can revert these effects.

Cytotoxic properties, glycolytic effects and high-resolution respirometry mitochondrial activities of Eriocephalus racemosus against MDA-MB 231 triple-negative breast cancer

IntroductionTriple-negative breast cancer (TNBC) represents a significant global health crisis due to its resistance to conventional therapies and lack of specific molecular targets. This study explored the potential of Eriocephalus racemosus (E. racemosus) as an alternative treatment for TNBC. The cytotoxic properties and high-resolution respirometry mitochondrial activities of E. racemosus against the MDA-MB 231 TNBC cell line were evaluated.MethodsHexane solvent and bioactive fraction extractions of E. racemosus were performed, while mass spectrometry-based metabolite profiling was used to identify the phytochemical constituents of the extracts. The extracts were further tested against MDA-MB 231 TNBC cells to determine their cytotoxicity. The mode of cell death was determined using flow cytometry. The activities of caspases 3, 8, and 9 were assessed using a multiplex activity assay kit. Glycolytic activity and High-resolution respirometry measurements of mitochondrial function in the MDA-MB 231 cell line were conducted using the Seahorse XFp and Oroboros O2K.ResultsMetabolite profiling of E. racemosus plant crude extracts identified the presence of coumarins, flavonoids, sesquiterpenoids, triterpenoids, and unknown compounds. The extracts demonstrated promising cytotoxic activities, with a half maximal inhibitory concentration (IC50) of 12.84 µg/mL for the crude hexane extract and 15.49 µg/mL for the bioactive fraction. Further, the crude hexane and bioactive fraction extracts induced apoptosis in the MDA-MB-231 TNBC cells, like the reference drug cisplatin (17.44%, 17.26% and 20.25%, respectively) compared to untreated cells. Caspase 3 activities confirmed the induction of apoptosis in both cisplatin and the plant crude extracts, while caspase 8 and 9 activities confirmed the activation of both the intrinsic and extrinsic apoptosis pathways. Increased levels of glycolytic activity were observed in the hexane crude extract. High-resolution respiratory measurements showed elevated mitochondrial activities in all mitochondrial states except for complex-IV activity.ConclusionThese findings support further exploration of E. racemosus as a potential therapeutic agent for TNBC, offering a promising avenue for the development of targeted treatments with minimal adverse effects.

The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer.

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

LncRNA HIF1A-AS2 promotes triple-negative breast cancer progression and paclitaxel resistance via MRPS23 protein

Dysregulation of lncRNAs is a critical factor in the migration and invasion of tumors. Here our study reveals that lncRNA HIF1A-AS2 is highly expressed in breast cancer tissues and various TNBC cell lines. Moreover, we present compelling evidence supporting the role of HIF1A-AS2 in promoting TNBC cell proliferation, metastasis, invasion, and resistance to paclitaxel treatment. Additionally, our transcriptome sequencing analysis identifies MRPS23 as a potential downstream target protein regulated by HIF1A-AS2 and knockdown of HIF1A-AS2 leads to decreased expression of MRPS23 in TNBC cells. Moreover, MRPS23 exhibits similar effects on enhancing cell proliferation, metastasis, invasion, and paclitaxel resistance in TNBC cells. Furthermore, downregulating HIF1A-AS2 suppresses the enhanced functionality observed in TNBC cells due to upregulated MRPS23 expression. These findings suggest that modulation of MRPS23 protein expression by HIF1A-AS2 may influence cellular processes and paclitaxel sensitivity in TNBC cells.

Isolation and chemical characterization of secondary metabolites from Gazania rigens and their antimicrobial and cytotoxic activities

Phytochemical study of Gazania rigens whole plant led to isolation and structural characterisation of six known compounds including lupeol (1), lupenone (2), β-sitosterol 3-O-β-glucopyranoside (3), dunalianoside D (4), 3,5-di-O-caffeoylquinic acid (5), and tachioside (6). Compounds (1–4, and 6) were isolated for the first time. The structures of the isolated compounds were elucidated using different spectroscopic analyses such as 1H,13C, and DEPT NMR analyses, besides HR-ESI-MS analysis. The isolated compounds and fractions were screened for in vitro antimicrobial activity against various bacterial and fungal strains. Only lupeol (1) exhibited a moderate antibacterial activity against K. pneumoniae with IC50 value of 19.05 µg/mL. The cytotoxic potential of the total ethanol extract and its derived fractions of G. rigens was evaluated against the MDA-MB-468 TNBC cell line using MTT assay. The petroleum ether, EtOAc and n-BuOH fractions exhibited a moderate inhibitory action with IC50 values of 22.42 ± 0.63, 25.55 ± 1.53, and 28.07 ± 0.63, respectively.

Cytotoxic potential of Curcuma caesia rhizome extract and derived gold nanoparticles in targeting breast cancer cell lines

Among all types of cancer, breast cancer is the most aggressive, as it is responsible for most of the cancer related death of women. Though several medical therapies are available, the scenario of curing such disease is not favorable. Therefore, there is an urgent need to find alternatives to deal with it. The knowledge of ethnopharmacy might give some better solution to mitigate such deadly diseases. Here, we are using the rhizome of Curcuma caesia Roxb. (Black turmeric), as well as gold nanoparticles (GNPs) synthesized with it to check their specific cytotoxic potentiality against breast cancer cell lines. In our study, ethanolic extract was used to evaluate the cytotoxic effect of the rhizome. GNPs were synthesized by using the same extract and characterized by UV–Vis spectroscopy (UV–Vis), Transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and Thermo gravimetric analysis (TGA). The TEM, XRD, FTIR and TGA results revealed the successful synthesis and capping of GNPs. The UV–Vis Spectrum showed a sharp and narrow absorption peak at 550 nm and HRTEM confirmed both the stability and successful synthesis of the nanoparticles. The MTT assay of the crude extract revealed strong cytotoxicity against breast cancer cell lines viz. MCF-7 (ER+) and MDA MB-231 (Triple Negative Breast Cancer, TNBC) by showing IC50 values as 15.70 ± 0.029 and 21.57 ± 0.031 μg/mL respectively. For extract mediated GNPs, the IC50 values were found to be 6.44 ± 0.045 and 5.87 ± 0.031μg/mL respectively in both breast cancer cell lines. As the IC50 value for GNPs was found to be much lower than that of crude extract, it indicates a higher efficiency of the GNP. However, both the rhizome extract and its mediated GNPs showed more toxicity towards MDA MB-231 (TNBC) cell lines. It was also observed that the GNPs showed more toxicity towards TNBC cell lines compared to the rhizome extract. No toxicity was found in case of other cell lines such as L 929 and HeLa for both crude extract as well as for GNPs. These observations suggests that both the crude rhizome extract and its derived GNPs exhibit selective cytotoxic potential against breast cancer cell lines, which might be exploited for target specific treatment. Moreover, with an understanding of the mechanism behind the GNPs therapeutic efficiency, it can be developed as a personalized therapy to treat such type of cancers.

Novel Thienopyrimidine-Hydrazinyl Compounds Induce DRP1-Mediated Non-Apoptotic Cell Death in Triple-Negative Breast Cancer Cells.

Apoptosis induction with taxanes or anthracyclines is the primary therapy for TNBC. Cancer cells can develop resistance to anticancer drugs, causing them to recur and metastasize. Therefore, non-apoptotic cell death inducers could be a potential treatment to circumvent apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induced non-apoptotic cell death in TNBC cells. These lead compounds were 15- to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to that of normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death, characterized by the absence of cellular shrinkage and the absence of nuclear fragmentation and apoptotic blebs. Although TPH104c and TPH104m induced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase the levels of cytochrome c and intracellular reactive oxygen species (ROS) and caspase activation, and cell death was not rescued by incubating cells with the pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). Furthermore, TPH104c and TPH104m significantly downregulated the expression of the mitochondrial fission protein, DRP1, and their levels determined their cytotoxic efficacy. Overall, TPH104c and TPH104m induced non-apoptotic cell death, and further determination of their cell death mechanisms will aid in the development of new potent and efficacious anticancer drugs to treat TNBC.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment.MethodsGene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays.ResultsBUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade.ConclusionsBUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

BUB1 Inhibition Sensitizes TNBC Cell Lines to Chemotherapy and Radiotherapy.

BUB1 is overexpressed in most human solid cancers, including breast cancer. Higher BUB1 levels are associated with a poor prognosis, especially in patients with triple-negative breast cancer (TNBC). Women with TNBC often develop resistance to chemotherapy and radiotherapy, which are still the mainstay of treatment for TNBC. Our previous studies demonstrated that a BUB1 kinase inhibitor (BAY1816032) reduced tumor cell proliferation and significantly enhanced radiotherapy efficacy in TNBC. In this study, we evaluated the effectiveness of BAY1816032 with a PARP inhibitor (olaparib), platinum agent (cisplatin), and microtubule poison (paclitaxel) alone or in combination with radiotherapy using cytotoxicity and clonogenic survival assays. BUB1 inhibitors sensitized BRCA1/2 wild-type SUM159 and MDA-MB-231 cells to olaparib, cisplatin, and paclitaxel synergistically (combination index; CI < 1). BAY1816032 significantly increased the radiation sensitization of SUM159 and MDA-MB-231 by olaparib, cisplatin, or paclitaxel at non-toxic concentrations (doses well below the IC50 concentrations). Importantly, the small molecular inhibitor of BUB1 synergistically (CI < 1) sensitized the BRCA mutant TNBC cell line HCC1937 to olaparib. Furthermore, the BUB1 inhibitor significantly increased the radiation enhancement ratio (rER) in HCC1937 cells (rER 1.34) compared to either agent alone (BUB1i rER 1.19; PARPi rER 1.04). The data presented here are significant as they provide proof that inhibition of BUB1 kinase activity sensitizes TNBC cell lines to a PARP inhibitor and radiation, irrespective of BRCA1/2 mutation status. Due to the ability of the BUB1 inhibitor to sensitize TNBC to different classes of drugs (platinum, PARPi, microtubule depolarization inhibitors), this work strongly supports the role of BUB1 as a novel molecular target to improve chemoradiation efficacy in TNBC and provides a rationale for the clinical evaluation of BAY1816032 as a chemosensitizer and chemoradiosensitizer in TNBC.

Stilbene-rich extract increases the cytotoxic effects of paclitaxel in hormone receptor-positive and triple-negative breast cancer spheroids

BackgroundTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks three receptors commonly found in breast cancer cells. It is associated with high mortality rates, and therefore, investigating therapies to increase survival rates is crucial. Plant-derived compounds are being explored as potential adjuvants for common chemotherapy drugs, such as paclitaxel (Pac). PurposeThe study aimed to evaluate the cytotoxic effect of a prenylated stilbene-rich extract (SRE) produced via a sustainable peanut hairy root culture system and observe its potential as an adjuvant for Pac in human triple-negative and hormone receptor-positive (HR+) breast cancer spheroids. The effects were compared to arachidin-1 (A-1), a cytotoxic prenylated stilbene present in the extract. MethodsSRE was produced from elicited peanut hairy root cultures. The extract was purified using chromatography techniques to obtain the prenylated stilbene arachidin-1 (A-1) with a purity of over 95 %. TNBC cell lines MDA-MB-231, MDA-MB-436, and HR+ breast cancer cell line MCF-7 were used to evaluate the cytotoxicity and apoptotic activity of SRE in comparison with A-1. Two-dimensional (2D) experiments were performed using cell viability assays and imaging microscopy. Three-dimensional (3D) spheroids cultures were established, and the impact of SRE alone and in combination with Pac on cell viability and caspase 3/7 activity was evaluated. ResultsSRE (10 μg/mL) inhibited cell proliferation by approximately 50 % in TNBC and MCF-7 cells in a time-dependent manner. Additionally, Annexin V FITC/PI staining revealed that SRE (10 μg/mL) induced more apoptosis than A-1 at the equimolar concentration (5 μM) in MDA-MB-231 cells. Combining SRE with Pac decreased spheroid cell viability and induced apoptosis by activating caspases 3 and 7 in TNBC and HR+ breast cancer spheroids. ConclusionsThese findings highlight the potential of SRE as a novel adjuvant for Pac chemotherapy in TNBC and HR+ breast cancer treatment.

Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry.

Research has shown a connection between vasculogenic mimicry (VM) and cancer progression. However, the functions of genes related to VM in the emergence and progression of TNBC have not been completely elucidated. A survival risk model was constructed by screening biomarkers using DESeq2 and WGCNA based on public TNBC transcriptome data. Furthermore, gene set enrichment analysis was performed, and tumor microenvironment and drug sensitivity were analyzed. The selected biomarkers were validated via quantitative PCR detection, immunohistochemical staining, and protein detection in breast cancer cell lines. Biomarkers related to the proliferation and migration of TNBC cells were validated via in vitro experiments. The findings revealed that 235 target genes were connected to the complement and coagulation cascade pathways. The risk score was constructed using KCND2, NRP1, and VSTM4. The prognosis model using the risk score and pathological T stage yielded good validation results. The clinical risk of TNBC was associated with the angiogenesis signaling pathway, and the low-risk group exhibited better sensitivity to immunotherapy. Quantitative PCR and immunohistochemistry indicated that the expression levels of KCND2 in TNBC tissues were higher than those in adjacent nontumor tissues. In the TNBC cell line, the protein expression of KCND2 was increased. Knockdown of KCND2 and VSTM4 inhibited the proliferation and migration of TNBC cells in vitro. In this study, three VM-related biomarkers were identified, including KCND2, NRP1, and VSTM4. These findings are likely to aid in deepening our understanding of the regulatory mechanism of VM in TNBC.

Abstract PO3-25-08: Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target

Abstract Background: Metabolic rewiring is a hallmark of cancer that is often mediated by alterations in metabolic isozyme expression. Isozymes that are overexpressed in cancer and catalyze rate limiting steps in key metabolic processes are potential therapeutic targets. We previously identified phosphoenolpyruvate carboxykinase 2 (PCK2) as cancer dominant isoform, required for maintaining high metabolic activity and proliferation of lung, prostate, and breast cancer (BC) cells in vitro and in vivo. No PCK2 specific small molecule inhibitors exist. Methods: mRNA expression levels were assessed in the TCGA data. PCK2 MISSION shRNAs (Millipore) were used to knock-down expression of PCK2 in BC cells. Alterations in metabolic flux caused by PCK2 down-regulation were examined via 13C-labeling and mass spectrometry using a SCIEX 5500 QTRAP and SelexION instrument for mobility separation of metabolites. The AtomNetTM (Atomwise Inc.) deep convolutional neural network structure-based drug design software was used to identify candidate small molecule PCK2 inhibitors which were tested in PCK1 and PCK2 in vitro enzyme activity assays. The lead compound was assessed for drug-like properties by QickPropV4.0.001w, and its growth inhibitory activity was tested in MDA-MB-321 and BT-549 cells. Results: Analysis of mRNA levels in TNBC samples revealed a significant decrease in PCK1 expression compared to normal tissues, while PCK2 expression remained elevated or even increased. Quantification of mRNA levels in TNBC cell lines confirmed the higher expression of PCK2 compared to PCK1. To assess functional significance of PCK2 expression, we downregulated PCK2 using shRNA constructs in TNBC cell lines. Western blot analysis confirmed efficient knockdown of PCK2 protein without affecting PCK1 expression. Clonogenic assays demonstrated that PCK2-depleted BT-549 and MDA-MB-231 cells exhibited impaired proliferation, with reduced colony number and size compared to control cells. Metabolic analysis of PCK2-depleted TNBC cells demonstrated decreased levels of glycolytic and TCA intermediates and reduced flux through pyruvate carboxylase, indicating altered metabolic pathways upon PCK2 depletion. To identify potential inhibitors of PCK2, virtual screening was conducted using compound structures. We used a PCK2 protein structure homology model and designated the 3-mercaptopicolinic acid-binding site of the PCK1 structure as the site of interest to perform virtual drug screening of 7 million diverse compounds. The first screen yielded 86 structures to be tested in vitro, of which 5 demonstrated &amp;gt; 16% PCK2 inhibition. The top hit (IC50=2.4µM) was used to refine a second round of in silico screen that yielded 87 second-round candidates that were assessed for PCK2 inhibition in vitro. 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate emerged as the lead compound. QickProp predicted favorable absorption, distribution, metabolism, and excretion properties, but relatively low cell permeability (apparent Caco-2 and MDCK2 cell permeabilities 18 nm/sec and 8 nm/sec, respectively). The specificity of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was tested in PCK1 and PCK2 enzymatic assays and revealed IC50 of 500nM on PCK1 and an IC50 of 3 nM on PCK2. We next tested the cell growth inhibitory effect of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on parental MDA-MB-231 and BT-549 cells and their PCK2 knockdown clones. Cell growth and viability significantly decreased in the MDA-MD-231 and BT-549 parental cells (the IC50 for MDA-MB-231 was 173 μM) and in shRNA non-targeting control cells, but not in the PCK2 depleted clones, demonstrating PCK2-dependent inhibitory effect. Conclusion: We identified 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate as a high affinity selective PCK2 enzyme inhibitor. This compound also has significant growth inhibitory activity in BC cell lines in vitro and represents a novel therapeutic lead compound. Citation Format: Alejandro Rios Hoyo, Naing Lin Shan, Hao-Kuen Lin, Gerson Espinoza Campos, Mostafa Ahmed, Sheila Umlauf, Rebecca Cardone, Yulia V. Surovtseva, Vignesh Gunasekharan, Lajos pusztai. Artificial Intelligence-based screening of small molecule inhibitors of phosphoenolpyruvate carboxykinase 2 (PCK2), a novel cancer therapeutic target [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-08.

Abstract PO5-06-11: RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis

Abstract Background: Ribosome is a complex structure in cells for protein synthesis. It has been considered always have a constant composition. Recent studies have revealed that the Ribosome composition of tumor cells is heterogeneous. The Ribosome protein composition, post-translational modification, rRNA modification, and rRNA variants can lead to the existence of specialized "onco-ribosomes" in tumor cells, which regulates the rate of translation and protein synthesis, and can affect the occurrence and development of tumors significantly. The abnormal expression of multiple Ribosome proteins in triple-negative breast cancer may be the target of its diagnosis and treatment, also, an independent factor in prognosis evaluation. Methods: To learn the expression levels of Ribosome biogenesis related genes in normal breast cells and abnormal breast cells with different pathological subtypes of breast cancer, the following studies have been done: 1. Bioinformatics analysis on TCGA database and single-cell sequencing data; 2. IHC detection on TNBC tissue and adjacent tissues; 3. Determine the expression level of RPL27A in TNBC cells, and analyze the correlation between the expression of RPL27A and clinical information of patients. To explore the effect of RPL27A on Ribosome biogenesis, protein synthesis and other signaling pathways through Proteomics and molecular biology experiments have been designed and tested. The effects of RPL27A on malignant biological behaviors such as proliferation, migration, infiltration, and clonal formation of TNBC cells were studied through cell biology experiments and mouse experiments. Results: The analysis of TCGA database and single-cell sequencing data showed that the RPL27A was significantly increased in TNBC group. Immunohistochemical experiments showed that RPL27A was highly expressed in TNBC tissue and was impressively positively correlated with tumor size, lymph node metastasis, and bone metastasis. There was significantly negatively correlated with overall survival (OS) and disease free survival (DFS). It can be an independent factor for evaluating the prognosis of TNBC. By knocking out RPL27A in TNBC cell line, label free proteomics analysis showed that Ribosome biogenesis, protein synthesis, cell cycle regulation and other signaling pathways were obviously down regulated. Cell biology experiments have implied that the high expression of RPL27A in TNBC promotes cell proliferation, enhances cell migration, infiltration, and cloning ability, leads to more malignant biological behavior of tumor cells. Further research demonstrates that the high expression of RPL27A in TNBC significantly activated the EIF3C signal, cause changes in the composition of Ribosome in TNBC cells, promoted the formation of "onco-ribosomes", and distinctly increased the rate of protein synthesis. The study of PDX mice and Organoid showed that the knock out of RPL27A significantly reduced the growth rate and transfer ability of TNBC. Conclusion: The obvious over expressing of RPL27A in TNBC, which can significantly promote the growth and metastasis of tumor cells by influencing the Ribosome biogenesis process. Citation Format: Weipeng Zhao, Jingyi Zhang, Ye Zhu, Xin Yang, Xichuan Li. RPL27A promotes the growth and metastasis of triple negative breast cancer by regulating Ribosome biogenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-11.

Abstract PO3-15-01: Overexpression of N-glycosylation related gene OST4 promotes chemotherapy resistance in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with high malignancy and poor outcome. Resistance to chemotherapy leads to treatment failure. How to overcome drug resistance is the key point in TNBC treatment. Previously, we initiated a prospective multicenter single-arm NeoPath clinical study (ClinicalTrial.gov identifier: NCT03907800) to assess the efficacy and safety of nab-paclitaxel combined with carboplatin in the neoadjuvant therapy of triple negative and HER2-positive early breast cancer. A total of 99 TNBC patients were enrolled and the pCR rate in TNBC was 33.78%. Samples before and after treatment were collected. RNA-sequencing results showed that OST4 was of high-expression in drug-resistant TNBC tumor tissues compared with chemo-sensitive samples. To verify the clinical significance of OST4 expression levels in TNBC, we tested the expression of OST4 by immunohistochemical technique on a tissue microarray including 87 TNBC cases treated with chemotherapy. We found that high expression of OST4 was associated with poor tumor differentiation, and patients with high OST4 expression had more relapse after chemotherapy. Ex-vivo proliferation assay and foci formation assay further confirmed that over-expression of OST4 promoted chemo-resistance, whereas down-regulating OST4 could reverse resistance in TNBC cell lines. As an oligosaccharyltransferase, it is reported that OST4 played parts in N-glycosylation. So we try to elucidate the relationship between N-glycosylation and drug resistance. Glycoproteomics results verified the existence of differential N-glycosylation atlas in drug-resistant tumor tissues compared with drug sensitive samples. NGS1 was identified as a potential target of OST4 by co-immunoprecipitation and mass spectrometry. In conclusion, high expression of OST4 could promote chemotherapy resistance in TNBC trough N-glycosylation regulation. Targeting OST4 may help reverse drug-resistance and improve therapeutic effect in TNBC. Citation Format: Wei Wang, Deyue Liu, Shuning Ding, Yan Fang, Jiayi Wu, Li Zhu. Overexpression of N-glycosylation related gene OST4 promotes chemotherapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-01.

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Abstract PO1-25-12: Metformin enhances response to chemotherapy combined with immunotherapy in a triple negative breast cancer in vivo model

Abstract Background Triple-negative breast cancer (TNBC) is a heterogeneous group comprising several well-defined molecular subtypes. Classical chemotherapy remains the standard of care for advanced TNBC, although blockade of the PD-1/PD-L1 axis has emerged as a promising therapeutic option to enhance antitumor immunity. Atezolizumab is an immune checkpoint inhibitor targeting PD-L1 that has been approved for use in combination with paclitaxel for the treatment of advanced TNBC. Metformin (MTF) is a prescribed drug for type 2 diabetes with well-tolerated side-effects and has been reported to have anticancer effects. Xenograft mouse models studies of breast, lung and prostate cancer, have reveal a synergistic effect of MTF when combined with paclitaxel, carboplatin or doxorubicin and has been reported to enhance the efficacy of immunotherapy. In this work we want to explore the effect of including MTF in a chemo-immunotherapy scheme using an in vivo model. Methods 1x106 EMT6 TNBC cell line (ATCC® CRL-2755™) were implanted subcutaneously in the back of immunocompetent BALB/c mice. Treatment began when tumours reached a volume size of 30-100 mm3. At that moment, mice received the drug, or drugs combinations, for 21 days. All drugs were administered intraperitoneally, except for MTF, which was given orally in drinking water at a concentration of 1g/L. The anti PD-L1 drug anti-hPD-L1-mIgG1 (mATZ, 300 mg/week) was administered twice per week and doxorubicin (DOXO) every three days (0.3 mg/Kg or 6 mg/kg). The primary outcome will be tumour response. Tumour size was measured using a digital caliper every day until mice were euthanized. For tumour volume determination (mm3), two measures were taken (length and width). Volume was calculated as (length x wide2)/2. We tested three different arms: DOXO(0,3mg/Kg)+m-ATZ, DOXO(0,3mg/Kg)+m-ATZ+MTF, and DOXO(6mg/Kg)+m-ATZ. Each arm included 10 mice. Differences in tumour volume and mice weight were assessed by Kruskall-Wallis analysis. Results EMT6 cells produced a tumour mass in twenty seven out of thirty mice. Follow-up in mice in the DOXO(0.3mg/Kg)+m-ATZ and DOXO(6mg/Kg)+m-ATZ arms was interrupted in two weeks due to extended tumour growth and quality of live criteria, respectively. Mice in the DOXO(0.3mg/Kg)+m-ATZ+MTF arm were followed for three weeks. Increase of tumoral volume was assessed at 14 days. Mean increase was 205, 1096 and 365 mm3in the DOXO(6mg/Kg)+m-ATZ, DOXO(0,3mg/Kg)+m-ATZ, and DOXO(0,3mg/Kg)+m-ATZ+MTF arms respectively, being the volume increase between DOXO(0,3mg/Kg)+m-ATZ treated mice significantly higher. Regarding mice, mean weight at the beginning of the treatments was 19.8 gr for the complete mice population, whereas mean weight after 14 days of treatment was 15.4, 20.9 and 21.7 gr in the DOXO(6mg/Kg)+m-ATZ, DOXO(0,3mg/Kg)+m-ATZ, and DOXO(0,3mg/Kg)+m-ATZ+MTF arms respectively, showing a significant decrease of body weight in mice receiving a high dose of DOX, while no differences associated to the inclusion of MTF in the treatment scheme was found. Conclusions MTF addition to a combination of chemotherapy and immunotherapy in the treatment of TNBC increased therapy efficacy, with no added side effects. These result paves the way to explore treatment schemes with lower chemotherapy doses, decreasing toxicity side effects. Citation Format: Pilar Zamora, Angelo Gamez Pozo, Luz Vega-Clemente, Elena Lopez-Camacho, Lucia Trilla-Fuertes, David Alonso-Martin, Juan Angel Fresno Vara, Mariano Garcia-Arranz, Enrique Espinosa. Metformin enhances response to chemotherapy combined with immunotherapy in a triple negative breast cancer in vivo model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-12.

Novel combinatorial autophagy inhibition therapy for triple negative breast cancers

BackgroundTriple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. MethodsImmunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. ResultsMetabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. ConclusionIn this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Abstract LB261: Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line

Abstract Triple-negative breast cancer (TNBC) is a lethal female cancer, of which paclitaxel resistance is a major factor limiting treatment outcomes, and continued unmet needs for more effective therapeutic strategies are arduous. The aim of this study is to establish a paclitaxel-resistant TNBC cell line and to characterize the underlying mechanism for potential strategies to overcome the resistance. The human TNBC cell line MDA-MB-231 was exposed to stepwise escalating paclitaxel concentration from 0.5 to 50 nM and a resistant cell line T50R was selected. The IC50 of paclitaxel was 2 and &amp;gt;100 nM in MDA-MB-231 and T50R cells respectively. However, T50R cells required 50 nM Taxol for proliferation. When cultured in the absence of paclitaxel, T50R cells arrested at mitotic stage with high levels of spindle abnormalities. Since induction of microtubule (MT) stabilization plays critical roles in paclitaxel’s potency, we thus measure cell MT polymerization and kinetochore-MT stability. The results showed that, compared to parental MDA-MB-231 cells, the interphase MTs were less polymerized and the kinetochore-MTs were less stable in T50R cells, indicating that the MT dynamic instability was significantly increased in T50R cells. Thus, the increased dynamic unstable MTs in T50R cells might contribute to drug requirement for cell growth and counteract with paclitaxel effects, and consequently lead to drug-resistant phenotype. Identification and interference of endogenous regulators involved in the increased MT instability in T50R cells might provide leads to overcome paclitaxel resistance. Citation Format: Ling-Huei Yih, Hsiao-Hui Kuo, Chieh-Ting Fang. Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line [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 LB261.