Expression In Breast Cancer Cells Research Articles

Abstract PO1-26-05: Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations

Abstract Background: CAR T cells for the treatment of solid tumor cancers has not yet achieved the same success as CAR T cells for treatment of blood cancers. Two of the hurdles that must be cleared for effective use of CAR T cells for solid tumor cancers are: 1) CAR T cell exhaustion; and 2) failure to recognize and kill low antigen expressing cancer cells. A promising approach to overcoming CAR T cell exhaustion, referred to as “1XX” was developed in the Sadelain Lab at MSKCC. Mutation of Tyrosines to Phenylalanine in ITAMs 2 and 3, of the CD3z signaling domain, restrict signaling to ITAM 1. This slowing down of signaling has been reported to be effective at eliminating or greatly reducing CAR T cell exhaustion in blood cancers [Feucht, et al 2019; Park et al 2022; Schoutrop, et al 2023]. Purpose: To evaluate the potential of the 1XX mutations to overcome CAR T cell exhaustion and inability to kill low antigen expressing cells in an animal model of breast cancer. Specifically, we tested the ability of three different CARs to eliminate human breast tumors that expressed variable levels of the target antigen, MUC1*, and their ability to suppress tumor recurrence over approximately 100 days. Methods: All three CARs were targeted to the tumor by the same antibody fragment, huMNC2, that recognizes MUC1*, which is the transmembrane cleavage product of MUC1 that functions as a potent growth factor receptor. The CARs are: 1) huMNC2-41BB-CD3z; 2) huMNC2-CD28-CD3z; and 3) huMNC2-CD28-1XX. Heterogeneous tumors expressing different levels of target antigen were made as follows. T47D breast cancer cells were engineered to express even more of the target, MUC1*, and were also engineered to fluoresce green. T47D wild type cells were engineered to fluoresce red. Heterogenous tumors consisting of 250,000 cells were implanted sub-cu into female NSG mice bearing 90-day estrogen pellets. The tumors comprised either 70% wild-type/30% overexpressing cells, or 85% wild-type/15% overexpressing cells or 92.5% wild-type/7.5% overexpressing cells. Tumor engraftment was verified by bioluminescence at Day 6 post implantation. There was a total of 150 animals, 5 animals per group. Animals were administered a single dose of CAR T cells into the tail vein on Day 7, wherein the Effector to Target ratio was either 10:1, 5:1 or 1:1. Tumor volume was measured weekly by Luciferase/Luciferin bioluminescence on an IVIS instrument. In addition, the red versus green fluorescence of the tumor was tracked periodically as an indicator of which cells, high or low antigen expressing, were being killed. Between Day 93 and Day 96, animals were sacrificed, cells were recovered from blood and spleen, recovered tumors were weighed, tumor cells dissociated and fluorescent images were captured to determine which cells escaped CAR T cell killing. Conclusions: The CAR with the 1XX mutations in CD3z, huMNC2-CD28-1XX, was much more effective at suppressing breast tumor recurrence than either CAR with wild-type CD3z. At sacrifice, significantly more CAR T cells were recovered from huMNC2-CD28-1XX than from huMNC2-41BB-CD3z or huMNC2-CD28-CD3z. At high dose, the CARs with wild-type CD3z effectively suppressed the high antigen expressing cells, but the recurrent tumors were essentially made up of the low antigen expressing cells that had escaped CAR T cell killing. At low CAR T cell dose, the CARs with wild-type CD3z appeared to become exhausted about 40 days post treatment, when tumors began to recur. Post sacrifice analysis of the recurrent tumors showed that they were made up of both high and low antigen expressing cells. huMNC2-CD28-1XX effectively killed both the high antigen and low antigen expressing cells as evidenced by the live fluorescent imaging and the post-sacrifice analysis of residual tumor. Citation Format: Cynthia Bamdad, Andrew Stewart, Benoit Smagghe, Mark Carter, Danica Walkley, Kevin Yi, Jac-Leen Nash, Michael Nash, Trevor Grant, Gregory Riley. Animal Study Compares CAR T cell Exhaustion & Ability to Kill Low Antigen expressing breast cancer cells among three CAR constructs including one with 1XX mutations [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 PO1-26-05.

Abstract PO4-15-03: Quantitative Measurement of TROP2 via Chromogenic Immunohistochemistry in Breast Cancer

Abstract Sacituzumab govitecan (SG) is an antibody-drug conjugate that targets human trophoblast cell-surface antigen 2 (TROP2), which is expressed in over 90% of breast cancer cases (Zaman et al, 2019). By delivering the cytotoxic SN-38 (topoisomerase I inhibitor) to TROP2 expressing breast cancer cells, SG showed promising antitumor activities in clinical trials and is now an approved treatment for triple-negative breast cancer (TNBC) and hormone receptor (HR)-positive HER2-negative breast cancer in the metastatic setting. Despite the prevalent expression of TROP2 in breast cancer, the objective response rates reported from clinical trials were about 30%, indicating the need for a diagnostic test that can identify which patients are likely to benefit from therapy. Although the drug is approved without a companion diagnostic assay, recent data from the TROPiCS-02 trial has shown that TROP2-low (H-score < 100) showed a non-significant hazard ratio for benefit from SG. In the same study, patients treated with SG with H-score >100 showed a significant hazard ratio for benefit compared to physician’s choice (Tolaney et al, ASCO 2023). This suggests that TROP2 expression level is associated with SG response and that a threshold for TROP2 expression level may aid patient selection. Although over 90% of breast cancer patients were considered TROP2-positive by IHC, the expression level of TROP2 was never quantitatively assessed in large breast cancer cohorts. Here we describe a quantitative chromogenic immunohistochemistry (IHC) assay with a cell line standard. Mass spectrometry was used to measure TROP2 peptide concentrations in the six standard cell lines, and the correlation between the mass spectrometry and IHC data for each cell line was then used to convert chromogenic signals to concentrations of TROP2 protein (fmol/mm2). The antibody used in this assay was rigorously validated, and the antibody concentration was optimized for the best signal-to-noise ratio. The optical density (OD) of chromogenic staining was measured using QuPath 0.4.3 (Qymia extension) by identifying the tumor area via object classifiers and manual editing and then calculating the area-normalized sum of OD. Collectively, this assay can measure up to 29.1 fmol/mm2 of TROP2. By applying this assay to two serial retrospective primary breast cancer cohorts from Yale University, we quantitatively measured TROP2 expression levels in 332 clinical cases. Not surprisingly, over 90% of cases showed some chromogenic signal, with a median TROP2 concentration of 2.1 fmol/mm2 and a maximum of 10.5 fmol/mm2. TROP2 expression levels showed no significant association with clinicopathologic characteristics including race, stage, BRCA mutation status, molecular subtype, HER2 IHC levels, and outcome. Further work is underway to optimize the assay toward the goal of determination of whether there is a quantitatively definable threshold for TROP2 expression below which patients are unlikely to benefit from SG or other TROP2-targeted therapies. Citation Format: Mengni He, Matthew Liu, Thazin Aung, Sneha Burela, Katherine Bates, Charles Robbins, David Rimm. Quantitative Measurement of TROP2 via Chromogenic Immunohistochemistry in 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 PO4-15-03.

Abstract PO5-04-06: Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation

Abstract Chimeric antigen receptor T cells (CARTs) targeting Her2 represent a promising approach for the treatment of patients with Her2 expressing breast cancers (BC). Her2 CARTs confront major challenges within the immune-suppressive tumor microenvironment (TME) of breast cancer, which inhibits sustained T cell proliferation and persistent antitumor activity. To bolster Her2 CARTs' expansion and persistence, we engineered a TNF-related apoptosis-inducing ligand receptor 2 (TR2) specific scFv connected to the endodomain of 4-1BB (TR2BB). TR2BB co-expressing Her2 CARTs exhibited enhanced antitumor potential against BC tumors by triggering myelod derived suppressor cell (MDSC) apoptosis, thereby remodeling the TME and promoting T cell proliferation. Despite better tumor control in mice receiving CAR.TR2BB T cells, recent trials have demonstrated the importance of incorporating a cytokine signal for optimal tumor control in patients with solid tumors. Thus, we hypothesized that we could further augment anti-tumor activity of Her2 CAR.TR2BB T cells against BC by incorporating a cytokine signal 3 through IL-15 or C7R. We engineered the CARTs to (1) ectopically produce IL-15, a crucial T-cell homeostasis and survival cytokine, and (2) express a constitutively active IL-7 receptor (C7R) that triggers IL-7 receptor signaling independently of ligand or the common receptor gamma chain (γc). Our repeat weekly tumor challenge assay revealed that CAR.TR2BB.IL15 T cells outperformed CAR.TR2BB.C7R and CAR.TR2BB in T cell proliferation. Both CAR.TR2BB.IL15 and CAR.TR2BB.C7R exhibited improved cytotoxic potential after three weeks of antigen stimulation compared to CAR.TR2BB alone in comparison with CAR.TR2BB alone. Furthermore, in our in vivo xenograft NSG mouse models implanted with Her2 expressing breast cancer cell line and MDSCs, CAR.TR2BB.IL15 T cells showed superior tumor control and improved survival rates. In summary, the addition of IL15 transgene to our TME targeting Her2 CARTs provides an advantage in terms of T cell persistence, proliferation, and tumor control in in vitro and in vivo models. These promising preclinical results propel us to apply this optimized Her2 targeting CART therapy in a phase I clinical trial for patients with metastatic Her2-expressing breast cancers. Citation Format: Diego Chamorro, Lauren Somes, Emily Madaras, Saisha Nalawade, Paul Shafer, Andres Mosquera, Mary Kathryn McKenna, Valentina Hoyos. Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation [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 PO5-04-06.

Abstract PO5-24-08: Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53

Abstract Background: Cancer cells are constantly exposed to harsh conditions like nutrient deprivation, hypoxia, and calcium imbalance leading to the induction of UPR. In its early phase, UPR aims to restore homeostasis while prolonged stress leads to induction of apoptosis. Thus, it is not surprising to observe that expression of the UPR components has been reported to both promote as well as suppress tumorigenesis. Studies have shown that Cation transport regulator homolog 1 (CHAC1) plays a dual role in cancer progression. The objective of the present study is to investigate the p53 mediated regulation of CHAC1 in breast cancer. Methods: We initially subjected a breast cancer tissue microarray (TMA) to immunohistochemistry (IHC) with p53 and CHAC1 specific antibody. We further did in vitro experiments like western blotting, qRT-PCR to see the expression of CHAC1 when wild type (WT) p53 was either induced using camptothecin (CPT) and Nutlin-3 in WT p53 expressing breast cancer cells or by the ectopic WT and mutant p53 transfection in null p53 breast cancer cells (MDA-MB-157). The Chromatin immunoprecipitation (ChIP) assay was done to find a potential binding site of p53 on CHAC1 promoter. Further, in vitro study was done to elucidate a potential role of CHAC1 in cell proliferation. Results: The IHC staining analysis revealed that there was no significant difference in the CHAC1 expression between high and low p53 staining tissue cores as high CHAC1 staining was observed in both cases. Further, in vitro analysis reinforced the findings of CHAC1 in TMAs as cell line studies showed that both forms of p53 had enhancing effect on CHAC1 levels. Upon induction of WT p53 and/or ectopic transfection of WT and mutant p53, we observed that CHAC1 levels were upregulated in a dose dependent manner. ChIP assay revealed that while WT p53 occupies the promoter region of the CHAC1 gene to induce its expression, mutant p53 may be inducing CHAC1 expression through an unknown mediator. These observations provide the basis that CHAC1 may be a novel transcriptional target of p53. Ectopic transfection of WT and mutant 53 showed opposite effect on cellular proliferation as evident from BrdU cellular proliferation assay in MDA-MB-157 cells. Further, we showed that CHAC1 enhanced breast cancer cell proliferation possibly via upregulation of PI3K/Akt/mTOR pathway and may likely be a player in angiogenesis as shown by in vitro angiogenesis assay. Conclusion: Taken together, the data from present study suggests that the effect of CHAC1 on cellular proliferation was associated with the status of p53 in breast cancer cells. The results suggest a plausible explanation for the differential behavior of CHAC1 in tumorigenesis. Citation Format: Vikrant Mehta, Harish Chander. Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53 [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 PO5-24-08.

LILRB2 promotes immune escape in breast cancer cells via enhanced HLA-A degradation.

Increased expression of leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) is associated with immune evasion in breast cancer (BC). The aim of this study to elucidate the role of LILRB2 in BC progression. LILRB2 expression in tumor tissues was detected by immunohistochemical staining. Human leukocyte antigen A (HLA-A) expression in BC cells was detected by Western blotting, and HLA-A ubiquitination was detected by immunoprecipitation and histidine pulldown assay. An in-situ tumor model was established in nude BALB/c mice to verify the role of LILRB2 in immune escape. Finally, the functions and potential mechanisms of LILRB2 in BC progression were explored using in silico data. LILRB2 was upregulated in BC tissues and cells, and correlated positively with poor prognosis. LILRB2 promoted BC progression by downregulating HLA-A expression. Mechanistically, LILRB2 facilitates the ubiquitination and subsequent degradation of HLA-A by promoting the interaction between the ubiquitin ligase membrane-associated ring finger protein 9 (MARCH9) and HLA-A. In syngeneic graft mouse models, LILRB2-expressing BC cells evaded CD8 + T cells and inhibited the secretion of cytokines by the cytotoxic CD8 + T cells. LILRB2 downregulates HLA-A to promote immune evasion in BC cells and is a promising new target for BC treatment.

PMM2 controls ERα levels and cell proliferation in ESR1 Y537S variant expressing breast cancer cells

PurposeMetabolic reprogramming in breast cancer (BC) subtypes offers potential personalized treatment targets. Estrogen receptor α (ERα)-positive BC patients undergoing endocrine therapy (ET) can develop ET-resistant metastatic disease. Specific mutations, like Y537S in ERα, drive uncontrolled cell proliferation. Targeting mutant receptor levels shows promise for inhibiting growth in metastatic BC expressing ERα variants. Additionally, metabolic reprogramming occurs in ERα Y537S mutant cells. Consequently, we conducted a screen to identify metabolic proteins reducing intracellular levels of ERα Y537S and inhibiting cell proliferation. MethodsNine metabolic proteins were identified in a siRNA-based screen, with phosphomannose mutase 2 (PMM2) showing the most promise. We measured the impact of PMM2 depletion on ERα stability and cell proliferation in ERα Y537S mutant cells. Additionally, we tested the effect of PMM2 reduction on the hyperactive phenotype of the mutant and its proliferation when combined with metastatic BC treatment drugs. ResultsPMM2 emerged as a significant target due to its correlation with better relapse-free survival, overexpression in ERα-positive tumors, and its elevation in ERα Y537S-expressing cells. Depletion of PMM2 induces degradation of ERα Y537S, inhibits cell proliferation, and reduces ERα signaling. Notably, reducing PMM2 levels re-sensitizes ERα Y537S-expressing cells to certain ET drugs and CDK4/CDK6 inhibitors. Mechanistically, depletion of PMM2 leads to a reduction in ESR1 mRNA levels, resulting in decreased ERα receptor protein expression. Furthermore, the reduction of PMM2 decreases FOXA1 levels, which plays a crucial role in ERα regulation. ConclusionsOur findings establish PMM2 as an innovative therapeutic target for metastatic BC expressing the ERα Y537S variant, offering alternative strategies for managing and treating this disease.

Chitosan nanoplatform for the co-delivery of palbociclib and ultra-small magnesium nanoclusters: dual receptor targeting, therapy and imaging.

Theranostic nanoparticles have gained significant attention in cancer diagnosis and therapy. In this study, estrone (ES) and folic acid (FA) functionalized single and dual receptor targeted theranostic chitosan nanoparticles were developed for breast cancer imaging and therapy. These nanoparticles (NPs) were loaded with palbociclib (PB) and ultra-small magnesium nanoclusters (UMN). The developed nontargeted theranostic NPs (PB-UMN-CS-NPs), estrogen receptor targeted theranostic NPs (PB-UMN-CS-ES-NPs), folate receptor targeted theranostic NPs (PB-UMN-CS-FA-NPs), and dual targeted theranostic NPs (PB-UMN-CS-ES-FA-NPs) have particle sizes of 178.4 ± 1.21 nm, 181.6± 1.35 nm, 185.1± 1.33 nm, and 198.2± 1.43 nm with surface charges of +19.02± 0.382 mV, +13.89±0.410 mV, +16.72±0.527 mV and +15.23±0.377 mV, respectively. Cytotoxicity studies on estrogen receptor (ER) and folate receptor (FR) expressing breast cancer cells revealed that dual-targeted theranostic NPs (PB-UMN-CS-FA-ES-NPs) were more effective, inhibiting cell growth by 54.17 and 42.23 times in MCF-7 and T-47D cells compared to free PB, respectively. Additionally, developed NPs were capable of inhibiting the cell cycle progression of MCF-7 cells from the G1 phase to the S phase more efficiently compared to free PB. Ultrasound and photoacoustic (USG/PA) imaging demonstrated that dual targeted theranostic NPs were capable of effectively reducing hypoxic tumor volume and significantly suppressing tumor vascularity compared to free PB, nontargeted, FR targeted and ER targeted NPs. Moreover, in vivo optical imaging demonstrated tumor specific accumulation of the dual-targeted theranostic NPs. Furthermore, in vitro hemocompatibility and histopathological studies confirmed the biocompatibility of developed nanoformulations.

Circ_0001982 aggravates breast cancer development through the circ_0001982-miR-144-3p-GSE1 axis.

This study was designed to explore the role of circ_0001982 in breast cancer (BC) development.Quantitative real-time polymerase chain reaction and western blot analysis assays were used to determine circ_0001982, miR-144-3p, and gse1 coiled-coil protein (GSE1) expression. Functional assays were performed to evaluate cell proliferation, apoptosis, migration, and invasion. The glycolysis was analyzed with commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assays were conducted to analyze the relationships among circ_0001982, miR-144-3p, and GSE1. A murine xenograft model assay was performed to determine circ_0001982-induced effects on BC cell tumor properties in vivo.Circ_0001982 expression was upregulated, but miR-144-3p was reduced in BC tissues and cells in comparison with normal breast tissues and normal human mammary epithelial cells. Circ_0001982 knockdown or miR-144-3p overexpression inhibited BC cell proliferation, glycolysis, migration and invasion, and promoted apoptosis. Circ_0001982 sponged miR-144-3p and negatively regulated miR-144-3p expression in BC cells. In addition, GSE1 was identified as a target mRNA of miR-144-3p. Ectopic GSE1 expression relieved circ_0001982 depletion-induced effects on BC cell tumor properties. Furthermore, circ_0001982 absence suppressed BC cell tumor properties in vivo.Circ_0001982 contributed to the BC cell tumor properties by regulating the miR-144-3p-GSE1 axis.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p<.05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Identification of differentially expressed miRNAs and mRNAs associated with the regulation of breast cancer via in silico and in vitro methods.

miRNA expressions are altered during development of breast cancer(BC). The aim of this study is to identify novel cancer-related miRNAs and pathways to understand the mechanisms of BC subtypes. GSE59247 dataset was downloaded from gene expression omnibus(GEO) database and analyzed with GEO2R software. The differential miRNA expressions in BC cells were evaluated by miRNome PCR array. Venn diagram was used to reveal co-differentially expressed miRNAs between GSE59247 dataset and miRNome array. Clinical prognostic significance of selected miRNAs was evaluated via Kaplan Meier curve. KEGG pathway enrichment analysis was performed to find miRNA targets and results were validated by TNM plot analysis and q-RT-PCR. TargetScan database was used to predict the association of miRNAs and 3'-untranslated regions of target genes and their expressions were visualized by human protein atlas database. Venn diagram analysis showed overlap of 11 miRNAs from in silico and in vitro analysis. KEGG analysis revealed 'Lysine Degradation Pathway' as the most significantly enriched targeted pathway. q-RT-PCR results confirmed that Lysine degradation pathway related genes SETD7, SETDB2, EHHADH, SETMAR, KMT2A and SUV39H2 were differentially expressed in BC cells. Target prediction analysis identified binding sites between miR-1323-5p and 3'-UTR of SETD7, miR-129-5p and 3'-UTR of EHHADH and miR-628-5p and 3'-UTR of SETDB2 mRNA. Notably, miR-1323-5p, miR-129-5p, and miR-628-5p are differentially expressed in BC and they bind to 3'UTR of critical genes of Lysine degradation pathway, namely SETD7, SETDB2 and EHHADH. These miRNAs might serve as potential diagnostic and prognostic biomarkers for progression.

Fusobacterium nucleatum-derived small extracellular vesicles facilitate tumor growth and metastasis via TLR4 in breast cancer

BackgroundThe contributive role of the microbiome in tumor progression has been reported in multiple studies, such as the Fusobacterium nucleatum (F. nucleatum) in breast cancer (BC). This study aimed to explore the role of F. nucleatum-derived small extracellular vesicles (Fn-EVs) in BC and preliminarily uncover the mechanism.MethodsTen normal and 20 cancerous breast tissues were harvested to investigate the gDNA expression of F. nucleatum and its relation with the clinical characteristics of BC patients. After isolating Fn-EVs by ultracentrifugation from F. nucleatum (ATCC 25,586), both MDA-MB-231 and MCF-7 cells were treated with PBS, Fn, or Fn-EVs, followed by being subjected to CCK-8, Edu staining, wound healing, and Transwell assays to detect their cell viability, proliferation, migration, and invasion. TLR4 expression in BC cells with diverse treatments was assessed by western blot. In vivo experiments were performed to verify its role in tumor growth and liver metastasis.ResultsThe F. nucleatum gDNA levels of breast tissues in BC patients were significantly higher than those in normal subjects, and positively associated with tumor size and metastasis. Fn-EVs administration significantly enhanced the cell viability, proliferation, migration, and invasion of BC cells, while knocking down TLR4 in BC cells could block these effects. Furthermore, in vivo study verified the contributive role of Fn-EVs in tumor growth and metastasis of BC, which might rely on its regulation of TLR4.ConclusionsCollectively, our results suggest that F. nucleatum plays an important role in BC tumor growth and metastasis by regulating TLR4 through Fn-EVs. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

Enhanced E6AP-mediated ubiquitination of ENO1 via LINC00663 contributes to radiosensitivity of breast cancer by regulating mitochondrial homeostasis

Radiotherapy has shown measurable efficacy in breast cancer (BC). Elucidating the mechanisms and developing effective strategies against resistance, which is a major challenge, is crucial. Mitochondria, which regulate homeostasis of the redox environment, have emerged as a radiotherapeutic target. However, the mechanism via which mitochondria are controlled under radiation remains elusive. Here, we identified alpha-enolase (ENO1), as a prognostic marker for the efficacy of BC radiotherapy. ENO1 enhances radio-therapeutic resistance in BC via reducing the production of reactive oxygen species (ROS) and apoptosis in vitro and in vivo through modulation of mitochondrial homeostasis. Moreover, LINC00663 was identified as an upstream regulator of ENO1, which regulates radiotherapeutic sensitivity by downregulating ENO1 expression in BC cells. LINC00663 regulates ENO1 protein stability by enhancing the E6AP-mediated ubiquitin-proteasome pathway. In BC patients, LINC00663 expression is negatively correlated with ENO1 expression. Among patients treated with IR, those who did not respond to radiotherapy expressed lower levels of LINC00663 than those sensitive to radiotherapy. Our work established LINC00663/ENO1 critical to regulate IR-resistance in BC. Inhibition of ENO1 with a specific inhibitor or supplement of LINC00663 could be potential sensitizing therapeutic strategies for BC.

Abstract P3-06-08: Turning tumor cells into antigen-presenting cells for cancer immunotherapy

Abstract Miguel Lopez-Lago1, Sagarika pachhal1, Charles L. Wiseman1, William V. Williams1. 1BriaCell Therapeutics Corporation, Philadelphia, PA Turning tumor cells into antigen-presenting cells for cancer immunotherapy Background: Therapeutic cancer vaccines are based on specific stimulation of the immune system using tumor antigens to elicit an antitumor response. We have been conducting clinical trials using the breast cancer cell line SV-BR-1-GM as a therapeutic vaccine. SV-BR-1-GM is a GM-CSF expressing breast cancer cell line with features of an antigen presenting cell (APC) owning to the expression of several immunomodulatory molecules, including MHC-I (HLA-A, B &amp; C) and MHC-II (HLA-DRB3 &amp; -DRA). Initial results in patients treated with irradiated SV-BR-1-GM cells, low dose cyclophosphamide and local IFNα suggest that patients that match SV-BR-1-GM at least at 1 HLA allele are more likely to derive clinical benefit. This clinical observation, together with the fact that SV-BR-1-GM cells can directly activate CD4+ T-cells in an antigen-specific HLA-restricted manner, as demonstrated by an in vitro antigen presentation assay (1), lead us to hypothesize that SV-BR-1 (the parent cell line) can function as APC. To further enhance direct antigen presentation to T-cells, SV-BR-1 cells have been genetically modified to express co-stimulatory molecules, immunomodulatory cytokines, and an extended repertoire of HLA alleles. Methods: To generate an off-the-shelf semi-allogeneic cell therapy covering most of the population, SV-BR-1 was genetically modified to express an extended repertoire of HLA alleles. Based on population analysis, four cell lines, each carrying two (2) HLA-A and two (2) HLA-DRB3/4/5 alleles, should produce at least a single match in 99% of the population, with a 92% match at Class I HLA-A alleles and a 98% match at Class II HLA-DRB3/4/5 alleles. SV-BR-1 was genetically modified using CRISPR/cas9 deletion of the endogenous HLA-A and HLA-DRB3 alleles and subsequent lentiviral mediated expression of alternative HLA-A and DRB3 alleles. To generate tumor cell lines with enhanced direct antigen presentation to T-cells, SV-BR-1 cells were genetically modified to express co-stimulatory molecules and immunomodulatory cytokines by using a lentiviral mediated expression system. Results: Following sequential CRISPR/Cas9 editing, the SV-BR-1 cells were cloned, and one clone selected (clone 17) for further engineering. Lack of expression of HLA-A and HLA-DRB3 was confirmed using flow cytometry and DNA sequencing. Clone 17 was subsequently transduced with 6 lentiviral vectors each expressing 2 genes under the control of separate promoters:CD86-IL12, CD80-HLA-DRA, 4-1BBL-IL7, GM-CSF-IFN, HLA-allele-1-HLA-allele-2 and HLA-DR-allele-1-HLA-DR-allele-2. Four cell lines were generated with different combinations of HLA alleles. Following selection, cells were evaluated by ELISA, flow cytometry and RT-PCR to confirm gene expression. Cell lines that secreted GM-CSF, IFN IL12, IL7 and expressed CD80, CD86, 4-1BBL, and both Class I and Class II HLA alleles are then transferred to GMP manufacturing. These modified breast cancer cell lines will be used in clinical studies designed to first evaluate the safety of intradermal inoculation with the irradiated cells and later combined with other agents to augment the immune response. Each patient will be treated with the cell line(s) that match them at least at one HLA allele. 1. Lacher M. D. et al, Front Immunol. 2018 May 15;9:776 Citation Format: Miguel Lopez-Lago, William Williams, Charles Wiseman. Turning tumor cells into antigen-presenting cells for cancer immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-08.

Deregulation of miR-375 Inhibits HOXA5 and Promotes Migration, Invasion, and Cell Proliferation in Breast Cancer.

Breast cancer (BC) is a highly aggressive tumour and one of the women's leading causes of cancer-related deaths in worldwide. MiR-375 overexpressed in BC cells, and its biological relevance is largely unknown. Here in, we explored the function of miR-375 in BC. MicroRNA-375 targets were predicted by online target prediction tools and found that HOXA5 is one of the potential targets. MTT assay was employed to assess the effect of miR-375 on cell proliferation, where migration and invasion transwell assays were applied to detect cell migratory and invasive ability. Besides, relative expression of miR-375 and HOXA5 was measured in BC and HEK-293 cells, and its downstream gene target expressions were evaluated by qRT-PCR and western blot. In this study, we found that miR-375 expression was higher in BC cell lines than in the HEK-293 cell line, whereas HOXA5 expression was significantly lower. Our study showed that exogenous inhibition of miR-375 promoted HOXA5 expression; on the contrary, miR-375 mimics down-regulated HOXA5 expression level. Knockdown of miR-375 expression in BC cells reduces cell proliferation, migration, and invasion by inverse correlation expression of HOXA5. Our findings associated that miR-375 accelerated apoptosis evasion, proliferation, migration, and invasion by targeting HOXA5. In addition, nucleolin interferes in miR-375 biogenesis while silencing of nucleolin significantly reduced miR-375 expression and increased HOXA5 expression in BC. Thus, miR-375/HOXA5 axis may represent a potential therapeutic target for BC treatment.

Phosphofructokinase 1 Platelet Isoform Enhances VEGF Expression in Part Through HIF-1α Up-regulation in Breast Cancer.

Phosphofructokinase 1 platelet isoform (PFKP) catalyzes a rate-limiting reaction in glycolysis. It is highly expressed in several tumors, including breast cancer (BC). It can regulate tumor progression through metabolic reprogramming and gene transcription. In addition, overexpression of vascular endothelial growth factor (VEGF) is commonly observed in BC, which is associated with poor prognosis. However, whether PFKP regulates VEGF expression in BC remains unknown. Thus, the aim of this study was to investigate whether PFKP could regulate VEGF expression in BC. We designed an in vitro study to investigate the role of PFKP in VEGF expression and angiogenesis using several experiments, including shRNA-mediated PFKP knock-down, RNAi-resistant PFKP restoration, qPCR, immunoblotting, luciferase reporter assay and tube formation assay. The clinical relationship between PFKP and VEGF was analyzed using The Cancer Genome Atlas (TCGA) database. PFKP expression was associated with VEGF expression in BC patients from the TCGA database. Importantly, PFKP played an essential role in the EGFR activation-induced VEGF expression in BC cells. Mechanistically, EGFR-phosphorylated PFKP Y64 played a critical role in AKT-mediated transcriptional expression of HIF-1α and subsequent VEGF transcription. Hence, PFKP expression played a role in human umbilical vein endothelial cells (HUVECs) tube formation by regulating VEGF expression in BC cells. These findings highlight a novel mechanism underlying the non-metabolic function of PFKP in VEGF expression in BC and provide a therapeutic potential of targeting PFKP in BC patients.

Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen‑induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL‑dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro‑apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro‑apoptotic action in an antiestrogen‑resistant breast cancer cell model. In addition, the present study identified a pro‑survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen‑resistant breast cancer cells survive EGFR targeting by pro‑survival autophagy. These pre‑clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro‑survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL‑dependent apoptosis.

Has_circ_0048764 promotes breast cancer progression by sponging miR-578 and regulating HMGA2 expression

Background Circular RNAs (circRNAs) function as important regulators in the progression of cancers. The role of circRNA_0048764 (circ_0048764) in the development of breast cancer (BC) remains inconclusive. This work investigates the biological function and molecular mechanism of circ_0048764 in BC. Methods Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression levels of circ_0048764, microRNA-578 (miR-578) and high mobility group AT-hook 2 (HMGA2) mRNA. The viability of BC cells was examined by cell counting kit 8 (CCK-8) assay. Besides, cyclin D1, proliferating cell nuclear antigen (PCNA) and HMGA2 expression levels were detected by western blot. The migrative and invasive capability of BC cells were probed by transwell assay. The relationships between miR-578 and circ_0048764 or HMGA2 3′-UTR were validated by dual-luciferase reporter gene assay. Results Circ_0048764 was highly expressed in BC tissues and cells, which was significantly associated with tumor size (≥2 cm), lymph node status (positive), and higher TNM stage of BC patients. Circ_0048764 depletion suppressed the proliferative, migrative, and invasive abilities of BC cells, which was rescued by transfection of miR-578 inhibitors. The binding sites were verified between circ_0048764 and miR-578. HMGA2 was identified to be a target of miR-578 in BC cells, and circ_0048764 positively regulated HMGA2 expression in BC cells via repressing miR-578. Conclusion Circ_0048764 promotes BC cell growth, migration and invasion via absorbing miR-578 and up-regulating HMGA2.

234 (PB-058) Poster - Neratinib could be effective as monotherapy or in combination with Trastuzumab in HER2 low expressing breast cancer cells and organoid model

The selection of breast cancer patients for anti-HER2 treatment is based on HER2 positivity defined by IHC (Immunohistochemistry) and/or in situ hybridization (ISH). However, there have been some discrepancies and ambiguity on HER2 classification, leading to several cut-off changes over the years and resulting in some of the patients with lower HER2 expressing tumours not offered anti-HER2 treatments. Several studies also suggested that a subset of the HER2 low breast tumours may respond to anti-HER2 treatments. The aim of this study is to understand the mechanisms of action and resistance of HER2 targeting treatments in HER2-low expressing breast cancer cells (BC cells). The HER2 expression of a panel of 8 breast cell lines was assessed by IHC, FISH, western blot and qRT-PCR and their responses to trasztuzumab was assessed based on HER2 expression. We also established a panel of patient-derived organoids (PDOs) from the cancer tissues obtained from breast cancer patients to assess the effects of anti-HER2 agents.

Effects of Combined Pentadecanoic Acid and Tamoxifen Treatment on Tamoxifen Resistance in MCF-7/SC Breast Cancer Cells.

Estrogen receptors are indicators of breast cancer adaptability to endocrine therapies, such as tamoxifen. Deficiency or absence of estrogen receptor α (ER−α) in breast cancer cells results in reduced efficacy of endocrine therapy. Here, we investigated the effect of combined tamoxifen and pentadecanoic acid therapy on ER−α−under−expressing breast cancer cells. Drug resistance gene expression patterns were determined by RNA sequencing analysis and in vitro experiments. For the first time, we demonstrate that the combined treatment of pentadecanoic acid, an odd−chain fatty acid, and tamoxifen synergistically suppresses the growth of human breast carcinoma MCF−7 stem cells (MCF−7/SCs), which were found to be tamoxifen−resistant and showed reduced ER−α expression compared with the parental MCF−7 cells. In addition, the combined treatment synergistically induced apoptosis and accumulation of sub−G1 cells and suppressed epithelial−to−mesenchymal transition (EMT). Exposure to this combination induces re−expression of ER−α at the transcriptional and protein levels, along with suppression of critical survival signal pathways, such as ERK1/2, MAPK, EGFR, and mTOR. Collectively, decreased ER−α expression was restored by pentadecanoic acid treatment, resulting in reversal of tamoxifen resistance. Overall, pentadecanoic acid exhibits the potential to enhance the efficacy of endocrine therapy in the treatment of ER−α−under−expressing breast cancer cells.