Triple-negative Breast Cancer Lines Research Articles

Abstract 1702: SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools

Abstract Single cell “omics” and lineage tracing analyses are valuable tools for studying heterogeneity and clonal dynamics. However, current clonal analyses do not easily enable live cell retrieval. That is a particular issue when further study of cells that were targeted for elimination or otherwise not selected during an experiment could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and retrieval of live cells. Single cell-derived clonal populations are generated from complex cell population, each is infected with a unique, heritable molecular barcode, and stocks of each individual barcoded clone (BC) are retained. BCs can be combined to create a version of the original cell population or to generate custom BC pools. We developed a highly sensitive, accurate, rapid, and inexpensive qPCR-based method for identifying and quantifying BCs in vitro and in vivo.We applied SunCatcher to various breast cancer cell lines, including murine Met1 triple negative breast cancer line. A heterogeneous pool comprised of 31 Met1 BCs reliably reproduced the proliferation rate, tumor-forming capacity, and cancer progression as the original parental cell line. Individual BCs, however, displayed significant differences in proliferation, tumor formation, and breast cancer cell hallmarks: cytokeratin 8/14, Zeb1 (mesenchymal marker), EpCAM (epithelial marker) PD-L1 (immune checkpoint protein) and MHC-I (antigen presentation). Five BCs consistently dominated the primary tumors at the ethical end point of 21 days: BC2, BC8, BC25, BC53, BC67. Interestingly, not all 5 BCs were the fastest growing clones, suggesting in vitro proliferation does not correlate with clone expansion in vivo. Two clones (BC43, 18) were not detected in the primary tumors at ethical endpoint, yet when injected alone, had tumor-forming capacity after a long latency (~60 days). Therefore, SunCatcher enabled identification of clones that are consequential for disease progression that would otherwise not be identified during pre-clinical experimentation. Using SunCatcher, we successfully detected Met1 spontaneous metastases as early as 3 weeks in the lung, long bone, and mandible. Long bone had the highest incidence and metastatic burden and were dominated by BC53 and 8 in 3/5 mice. Lung had lower metastatic burden and BC53 was the dominant clone in 4/4 mice. BC8 was exclusive to bone, suggesting site-specific metastasis capacity for different tumor clones. SunCatcher is a reliable, inexpensive, and rapid clonal barcoding method that enables deeper understanding of heterogeneity and clonal dynamics. We identified and analyzed BCs that were responsible for cancer progression, and, for the first time, could analyze BCs that were negatively selected during cancer progression. We envision that SunCatcher will be a useful cancer research tool. Citation Format: Qiuchen Guo, Milos Spasic, Adam Maynard, Gregory Goreczny, Amanuel Bizuayehu, Jessica Olive, Sandra McAllister. SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1702.

Abstract 2637: Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines

Abstract The Triple-Negative Breast Cancer (TNBC) subtype is a breast cancer subpopulation whose phenotype lacks estrogen and progesterone growth receptors, and lacks HER2 amplification. This contrasts with the Luminal and HER2+ subpopulations for which clinical precision therapies exist to target cancer proliferation and improve prognoses. Consequently, the discovery of new targets is necessitated by the TNBC subtype, which has the poorest patient outcomes. Our studies have identified MAP3K19 as one such potential target. MAP3K19 an understudied protein kinase with poorly characterized physiological functions outside of its roles in the Mitogen-Activated Protein Kinase (MAPK) signaling cascade. Broadly, the MAPK signaling has been associated with cell proliferation and differentiation in pathways both intracellular and extracellular, which in the context of cancer cell biology warrants investigation into cancer cell behavior as well as the associated tumor microenvironment (TME). The Published Kinase Inhibitor Set (PKIS) is an open-access drug panel designed by GlaxoSmithKline to identify novel kinases. Preliminary PKIS cell-viability screening identified GSK1440913, GW494601, and GSK907232A as compounds with the most adverse effects on cell phenotype and growth in TNBC lines MDA-MB-231 and PDX-derived TU-BcX-49S. Analysis of common targets revealed MAP3K19 a common target, suggesting its importance in cancer cell growth and proliferation. AT-9283, a more specific MAP3K19 inhibitor, was used to support the initial results. Treatment with AT-9283 resulted in a dose-dependent killing effect. Furthermore, MAP3K19 inhibition resulted in decreased EMT gene signatures by qPCR. To summarize, MAP3K19 was identified by the PKIS screen as a major kinase in TNBC. Treatment with AT-9283, a more specific MAP3K19 inhibitor, resulted in cellular death and reversal in EMT gene signature by qPCR. Future directions for this project involve genetic knock-down and overexpression of MAP3K19 to evaluate its roles in biology and its signaling mechanisms. Citation Format: Khoa Nguyen, Thomas Cheng, Madlin Alzoubi, Steven Elliott, Sean Lee, David Drewry, Bridgette Collins-Burow, John Brognard, Matthew Burow. Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2637.

Abstract P1-08-20: In silico analysis of a novel mathematical model integrating in vitro and in vivo imaging data reveals driving mechanisms of breast cancer response to NAT for personalized tumor forecasting

Abstract The early assessment of neoadjuvant therapy (NAT) response in triple-negative breast cancer (TNBC) would enable a treating oncologist to adjust a therapeutic plan of a non-responding patient, and thereby enhance outcomes while preventing unnecessary toxicities. To address this challenge, we propose leveraging personalized, in silico forecasts of tumor response to therapeutic regimens via a mechanistic mathematical model calibrated with patient-specific longitudinal magnetic resonance imaging (MRI) data acquired early during NAT. Here, we focus on identifying the driving mechanisms involved in the model formulation through a global sensitivity analysis. Our model describes the dynamics of TNBC cell density as a combination of mobility, which is formulated as a diffusion process constrained by local tumor-induced mechanical stress, and net proliferation, which is represented with a logistic term. To model TNBC response to NAT drug combinations, we adjust the tumor cell proliferation rate with a recent pharmacodynamic model, MuSyC, which also accounts for synergy of potency and efficacy. Tumor cell density is estimated from diffusion-weighted MRI data, while tissue mechanical properties are defined from segmented contrast-enhanced T1-weighted MRI data. To model the heterogeneous intratumoral delivery of drugs, we use perfusion maps estimated from dynamic contrast-enhanced MRI data. NAT drug pharmacokinetics are approximated with a linear model, which reasonably represents their temporal decay during NAT. Sobol’s method is used for the global sensitivity analysis of the NAT response of two different tumors (one well-perfused and one poorly-perfused) on a 3D, tissue-scale domain. This allows us to assess the total effect (ST) of each model parameter on tumor volume and global cellularity. Here, we focus on two standard NAT regimens: doxorubicin plus cyclophosphamide, and paclitaxel plus carboplatin. The parameter space is constructed by integrating three approaches. First, we use prior patient-specific in silico estimates of tumor cell mobility and proliferation. Second, we experimentally constrain the parameters accounting for potential synergistic drug activity by using time-resolved, high-throughput, automated microscopy assays that capture drug-induced changes in proliferation rates of various TNBC lines (HCC1143, SUM149, MDAMB231, and MDAMB468; perfosfamide was used in lieu of the pro-drug cyclophosphamide). Third, we scale the resulting in vitro parameter ranges to be clinically-relevant through an in silico study with our mechanistic model. Our results show that out of the 15 model parameters considered in the sensitivity analysis only a minority exhibited a driving role (ST > 0.1) in representing the dynamics of TNBC response to NAT, namely: the baseline tumor cell proliferation rate along with the effect and ratio of peak concentration to EC50 of doxorubicin, paclitaxel, and carboplatin. The other parameters have a marginal effect and can thus be fixed to any value within the parameter space. We select these constant parameters such that they contribute to simplifying the model formulation, leading to a surrogate reduced model. We further show that the reduced and the original model produce distributions of tumor volume and global cellularity that are in remarkable agreement both qualitatively (Dice similarity coefficient > 0.90 and > 0.94, respectively) and parameter-wise (concordance correlation coefficient > 0.85 and > 0.89, respectively). Thus, we conclude that our reduced model constitutes a feasible surrogate for future clinical calibration-forecasting studies, thereby facilitating personalized NAT response forecasting with patient-specific imaging datasets acquired in vivo. Citation Format: Guillermo Lorenzo, Angela M. Jarrett, Christian T. Meyer, Darren R. Tyson, Vito Quaranta, Thomas E. Yankeelov. In silico analysis of a novel mathematical model integrating in vitro and in vivo imaging data reveals driving mechanisms of breast cancer response to NAT for personalized tumor forecasting [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-20.

DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer.

Although chemotherapies kill most cancer cells, stem cell-enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1- populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1- cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1- cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1-. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1- cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1- populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell-enriched TNBC.

Involvement of Mitochondrial Mechanisms and Cyclooxygenase-2 Activation in the Effect of Desethylamiodarone on 4T1 Triple-Negative Breast Cancer Line.

Novel compounds significantly interfering with the mitochondrial energy production may have therapeutic value in triple-negative breast cancer (TNBC). This criterion is clearly fulfilled by desethylamiodarone (DEA), which is a major metabolite of amiodarone, a widely used antiarrhythmic drug, since the DEA previously demonstrated anti-neoplastic, anti-metastasizing, and direct mitochondrial effects in B16F10 melanoma cells. Additionally, the more than fifty years of clinical experience with amiodarone should answer most of the safety concerns about DEA. Accordingly, in the present study, we investigated DEA’s potential in TNBC by using a TN and a hormone receptor positive (HR+) BC cell line. DEA reduced the viability, colony formation, and invasive growth of the 4T1 cell line and led to a higher extent of the MCF-7 cell line. It lowered mitochondrial transmembrane potential and induced mitochondrial fragmentation. On the other hand, DEA failed to significantly affect various parameters of the cellular energy metabolism as determined by a Seahorse live cell respirometer. Cyclooxygenase 2 (COX-2), which was upregulated by DEA in the TNBC cell line only, accounted for most of 4T1’s DEA resistance, which was counteracted by the selective COX-2 inhibitor celecoxib. All these data indicate that DEA may have potentiality in the therapy of TNBC.

Auger Emitter Conjugated PARP Inhibitor for Therapy in Triple Negative Breast Cancers: A Comparative In-Vitro Study.

Simple SummaryTriple negative breast cancer (TNBC) is an aggressive subtype of breast cancer, with a high recurrence rate. Since treatment of BRCAmut TNBC patients with PARP inhibitor (PARPi), targeting the nuclear protein PARP1, shows varied responses, its therapeutic efficacy is currently evaluated in combination with chemotherapy. Auger emitters (AEs) are radionuclides that can cause DNA damage when delivered close to the DNA. Due to the nuclear location of PARP1, radiolabelling of PARPi with AEs provide an efficient nuclear delivery mechanism. This study shows the radiosynthesis of an AE radiolabelled PARPi ([125I]-PARPi-01) and its therapeutic effect as monotherapy or in combination with chemotherapeutics in a panel of TNBC cell lines. We found that [125I]-PARPi-01 efficiently induces DNA damage with therapeutic effect irrespective of BRCA mutation. All responsive cell lines have homologous recombination deficiency. Short pretreatment with doxorubicin significantly reduces clonogenic survival of both responsive and resistant cell lines.PARP1 inhibitors (PARPi) are currently approved for BRCAmut metastatic breast cancer, but they have shown limited response in triple negative breast cancer (TNBC) patients. Combination of an Auger emitter with PARPis enables PARP inhibition and DNA strand break induction simultaneously. This will enhance cytotoxicity and additionally allow a theranostic approach. This study presents the radiosynthesis of the Auger emitter [125I] coupled olaparib derivative: [125I]-PARPi-01, and its therapeutic evaluation in a panel of TNBC cell lines. Specificity was tested by a blocking assay. DNA strand break induction was analysed by γH2AX immunofluorescence staining. Cell cycle analysis and apoptosis assays were studied using flow cytometry in TNBC cell lines (BRCAwt/mut). Anchorage independent growth potential was evaluated using soft agar assay. [125I]-PARPi-01 showed PARP1-specificity and higher cytotoxicity than olaparib in TNBC cell lines irrespective of BRCA their status. Cell lines harbouring DNA repair deficiency showed response to [125I]-PARPi-01 monotherapy. Combined treatment with Dox-NP further enhanced therapeutic efficiency in metastatic resistant BRCAwt cell lines. The clonogenic survival was significantly reduced after treatment with [125I]-PARPi-01 in all TNBC lines investigated. Therapeutic efficacy was further enhanced after combined treatment with chemotherapeutics. [125I]-PARPi-01 is a promising radiotherapeutic agent for low radiation dosages, and mono/combined therapies of TNBC.

Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumour cell (DTC) dormancy in this organ is limited. Here, using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micro-metastatic state as the rate limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein (YAP), thereby sequestering it from the nucleus and preventing its pro-metastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.

Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

Triple negative breast cancer (TNBC) cells are generally more invasive than estrogen receptor-positive (ER + ) breast cancer cells. Consistent with the importance of activator protein 1 (AP1) transcription factors in invasion, AP1 activity is much higher in TNBC lines than ER + lines. In TNBC cells, robust AP1 activity is facilitated by both ERK and p38MAPK signaling pathways. While ERK signaling pathway regulates AP1 activity by controlling the abundance of AP1 transcription factors, p38MAPK signaling pathway does it by enhancing AP1 binding to AP1 sites without altering their abundance. Here, we show that p38MAPK regulation of AP1 activity involves both MAPKAPK2 (MK2) and JAB1, a known JUN-binding protein. MK2 not only interacts with JAB1 but also directly phosphorylates JAB1 at Ser177 in TNBC cells. Interestingly, Ser177 phosphorylation does not affect JAB1 and JUN interaction. Instead, interfering with p38MAPK signaling pathway or introducing an S to A point mutation at Ser177 of JAB1 reduces JUN recruitment to the AP1 sites in cyclin D1, urokinase plasminogen activator (uPA) and uPA receptor promoters. Moreover, knockdown of JAB1 diminishes >60% of AP1 transcriptional activity in TNBC cells. Taken together, these results indicate that MK2-mediated phosphorylation of JAB1 facilitates JUN recruitment to AP1 sites, thus augmenting AP1 activity. In line with the role of JAB1 in AP1 activity, silencing JAB1 leads to dramatic reduction in TNBC cell growth, in vitro invasion and in vivo tumor outgrowth. This study suggests that the p38MAPK-MK2 signaling pathway promotes TNBC tumorigenesis by sustaining robust AP1 activity.

Abstract 2715: Tamoxifen suppresses brain metastasis of estrogen receptor deficient breast cancer by skewing microglia polarization and enhancing their immune functions

Abstract Brain metastasis of breast cancer exhibits exceedingly poor prognosis, and both triple negative and Her2+ subtypes have the highest incidence of brain metastasis. Although estrogen blockers are considered to be ineffective for their treatment, recent evidence indicates that estrogen blockade using tamoxifen showed certain efficacy. However, how estrogen affects brain metastasis of triple negative breast cancer (TNBC) remains elusive. The communications between neoplastic cells and microglia, especially M2 type pro-tumor microglia, affect brain metastasis progression. Therefore, we hypothesize that estrogen skews microglial polarization to M2 phenotype which promotes brain metastasis. We found that M2 microglia were abundantly infiltrated in brain metastatic lesions of pre-menopausal TNBC patients. To test the effect of estrogen on brain metastasis, we transplanted the SKBrM brain metastatic cell TNBC line into nude mice by intra-cardiac injection followed by with or without 17β-estradiol (E2) treatment. We found that brain metastasis-free survival was decreased, while infiltration of M2 microglia was increased in E2-treated group. Blocking estrogen signaling either by tamoxifen treatment or surgical resection of ovaries suppressed M2 microglial polarization and brain metastasis. To test the effect of E2-driven microglia on tumor progression, brain metastatic cells were treated with conditioned medium (CM) generated from microglia that were treated with or without E2. We found that CM promoted cell growth and increased the population of cancer stem cells. Blocking E2-mediated M2 polarization by tamoxifen suppressed M2 polarization and decreased the secretion of C-C motif chemokine ligand 5, resulting in significant suppression of brain metastasis. To examine the mechanism by which estrogen suppresses anti-tumor function of microglia, we measured the expression of “do not eat me” signal, SIRP on microglia and CD47 on tumor cells. When SKBrM and microglial cells were treated with E2, we found that the protein expression of SIRP and CD47 were up-regulated on microglia and SKBrM, respectively, by E2 treatment. Furthermore, we used flow cytometry to detect the phagocytic ability of microglia by co-culturing SKBrM with microglia. We found that the phagocytic activity of microglia was significantly decreased upon E2 treatment. In contrast, tamoxifen restored E2-suppressed phagocytic ability. In conclusion, our results show that estrogen promotes brain metastasis by skewing polarity of M2 microglia and inhibiting their phagocytic ability, while tamoxifen suppresses brain metastasis by blocking M2 polarization of microglia and increasing their anti-tumor phagocytic ability. Our results also highlight a potential therapeutic utility of tamoxifen for treating brain metastasis of hormone receptor deficient breast cancer. Citation Format: Shih-Ying Wu. Tamoxifen suppresses brain metastasis of estrogen receptor deficient breast cancer by skewing microglia polarization and enhancing their immune functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2715.

MYBL1 Knockdown in a Triple Negative Breast Cancer Line: Evidence of Down-Regulation of MYBL2, TCF19 and KIF18b Expression

Purpose: The MYBL1 gene is a strong transcriptional activator, associated with cell cycle signaling and differentiation. Data show the gene is overexpressed in triple negative breast cancers. Considering the possibility that MYBL1 might be involved in events associated with the pathogenesis of these cancers, we sought to identify genes associated with MYBL1 expression in triple negative breast cancer. Methods: shRNA lentiviral knockdown was used to down-regulate the MYBL1 gene. Microarray analyses were used to identify genes either directly or indirectly affected by targeting MYBL1 knockdown. Data analyses was performed utilizing Affymetrix TAC 4.0, Chip X transcription factor analyses, Target Scan miRNA analyses, and STRING analyses was used to determine protein: protein interaction and pathway analyses. Web Gestalt and Gene Ontology were used to determine pathway and gene-set enrichments. Publicly available patient and cell line datasets were retrieved and processed using resources available in Gene Expression Omnibus and Oncomine. The polymerase chain reaction and western analyses were used to determine transcript and protein levels, respectively. Results: Knockdown of MYBL1 in a triple negative breast cell line led to down-regulation of MYBL2, TCF19, KIF18b along with an enrichment of cell cycle signaling genes. Gene expression analyses show that MYBL1, MYBL2, TCF19 and KIF18b display a similar pattern of expression in breast cell lines and many of the archival patient datasets examined. Conclusion: TNBC is a heterogeneous subtype, so these data suggest that cancers that over-express MYBL1, express MYBL2, TCF19 and KIF18b. Bioinformatic analyses suggest MYBL1 regulates MYBL2 which leads to regulation of TCF19 and KIF18b.

CXCL1 Clone Evolution Induced by the HDAC Inhibitor Belinostat Might Be a Favorable Prognostic Indicator in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer due to its lack of treatment options. Patients with TNBC frequently develop resistance to chemotherapy. As epigenetic-based antineoplastic drugs, histone deacetylase inhibitors (HDACis) have achieved particular efficacy in lymphoma but are less efficacious in solid tumors, and the resistance mechanism remains poorly understood. In this study, the GSE129944 microarray dataset from the Gene Expression Omnibus database was downloaded, and fold changes at the transcriptome level of a TNBC line (MDA-MB-231) after treatment with belinostat were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the critical biological processes. Construction and analysis of the protein-protein interaction (PPI) network were performed to screen candidate genes related to cancer prognosis. A total of 465 DEGs were identified, including 240 downregulated and 225 upregulated genes. The cytokine-cytokine receptor pathway was identified as being significantly changed. Furthermore, the expression of CXCL1 was implicated as a favorable factor in the overall survival of breast cancer patients. With in vitro approaches, we also showed that belinostat could induce the expression of CXCL1 in another 2 TNBC cell lines (BT-549 and HCC-1937). We speculate that belinostat-induced CXCL1 expression could be one of the results of the stress clone evolution of cells after HDACi treatment. These findings provide new insights into clone evolution during HDACi treatment, which might guide us to a novel perspective that various mutation-targeted treatments should be implemented during the whole treatment cycle.

Effect of Oxaliplatin, Olaparib and LY294002 in Combination on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) has a poor prognosis as the therapy has several limitations, most importantly, treatment resistance. In this study we examined the different responses of triple-negative breast cancer line MDA-MB-231 and hormone receptor-positive breast cancer line MCF7 to a combined treatment including olaparib, a poly-(ADP ribose) polymerase (PARP) inhibitor, oxaliplatin, a third-generation platinum compound and LY294002, an Akt pathway inhibitor. We applied the drugs in a single, therapeutically relevant concentration individually and in all possible combinations, and we assessed the viability, type of cell death, reactive oxygen species production, cell-cycle phases, colony formation and invasive growth. In agreement with the literature, the MDA-MB-231 cells were more treatment resistant than the MCF7 cells. However, and in contrast with the findings of others, we detected no synergistic effect between olaparib and oxaliplatin, and we found that the Akt pathway inhibitor augmented the cytostatic properties of the platinum compound and/or prevented the cytoprotective effects of PARP inhibition. Our results suggest that, at therapeutically relevant concentrations, the cytotoxicity of the platinum compound dominated over that of the PARP inhibitor and the PI3K inhibitor, even though a regression-based model could have indicated an overall synergy at lower and/or higher concentrations.

Abstract PS18-39: Estrogen receptor beta agonists for triple negative breast cancer

Abstract Background:Triple negative breast cancer (TNBC) represents about 10-15% of all breast cancers. Given the lack of targeted therapies, chemotherapy and immunotherapy are the only treatment options. The five-year survival rate of TNBC patients is approximately 15% less than other forms of breast cancer. Therefore, there is an urgent need to develop novel effective therapeutics for TNBC.Material and Methods:RNA isolated from human immortalized mammary epithelial MCF10A, and various TNBC cell lines was DNase treated and quantitative RT-PCR (qRT-PCR) performed using gene-specific primers spanning exon-exon junctions that include introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. Immuno-blot analyses were performed on lysates prepared from cells in log phase of growth. Proteins were detected by Enhanced Chemi-Luminescence (ECL) method. Viability of cells treated with ERβ specific agonists was determined by using CellTiter-Glo® 2.0 assay. MB231 cells were treated with ERβ agonists following transfection with pcDNA3 (vector) or Flag-tagged ESR2 plasmids. Western blot analyses using specific antibodies and qRT-PCR were performed with the protein extracts and RNA isolated from the cells, respectively. Results:Our experiments demonstrate that ESR2 (ERβ) is differentially expressed and its level of expression is about 3 to 22-fold higher in TNBC cell lines tested than MCF10A. We have shown that viability of estrogen receptor (ER) positive breast cancer cell lines can be significantly inhibited by selective activation of ERβ. A highly selective ERβ agonist has been invented in the Drug Development Institute at the Ohio State University. Treatment of TNBC lines with these ERβ agonists exhibited a dramatic reduction of cell viability. While comparable IC50 values were observed with our in house and commercially available compounds, TNBC had 2-3-fold lesser IC50 values than ER-positive cell lines. Increased expression of FOXO3 and c-Myc1 and decreased expression of c-Myc2 isoform was noted in ERβ overexpressing MB231 cells upon treatment with ERβ agonists.Conclusion:Our results demonstrate that treatment with highly selective ERβ agonists could be an effective therapeutic strategy for TNBC patients. To improve and develop novel therapeutics for TNBC understanding the mechanism of action of these compounds will be crucial. Citation Format: Jharna Datta, Natalie Willingham, Jasmine Manouchehri, Joel David, Mirisha Sheth, Bhuvaneswari Ramaswamy, Ramesh Ganju, Mathew A Cherian. Estrogen receptor beta agonists for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-39.

Liquiritigenin decreases tumorigenesis by inhibiting DNMT activity and increasing BRCA1 transcriptional activity in triple-negative breast cancer.

As a selective estrogen receptor β agonist, the natural flavonoid liquiritigenin reportedly inhibits invasiveness of breast cancer cells, but its specific role and mechanism remain largely unclear. In this study, cells from the triple negative breast cancer lines MDA-MB-231 and BT549 were incubated with different concentrations of liquiritigenin. The results indicated that low concentrations had no significant cytotoxic effect, whereas high concentrations decreased viability of both MDA-MB-231 and BT549 cells. Liquiritigenin treatment also resulted in increased apoptosis and enhanced Caspase3 activity. After liquiritigenin treatment, we observed decreased invasive and migratory capacities of cells, as well as upregulated E-cadherin and downregulated N-cadherin, vimentin, and MMP9. Interestingly, liquiritigenin increased the mRNA and protein expression of breast cancer 1 (BRCA1). It also increased p21 and growth arrest and DNA-damage-inducible 45 alpha (GADD45A) levels, accompanied by decreased cellular DNA methyltransferase (DNMT) activity and downregulation of DNMT1, DNMT3a, and DNMT3b. These findings suggest that liquiritigenin can inhibit malignant behavior of triple negative breast cancer cells by inhibiting DNMT activity and increasing BRCA1 expression and its transcriptional activity. Liquiritigenin thus may be a promising candidate for the treatment of breast cancer.

Differential Glucocorticoid-Dependent Regulation and Function of the ERRFI1 Gene in Triple-Negative Breast Cancer.

Glucocorticoids (GCs; eg, hydrocortisone [CORT]) are routinely used as chemotherapeutic, anti-emetic, and palliative agents in breast cancer (BCa) therapy. The effects of GC signaling on BCa progression, however, remain a contentious topic as GC treatment seems to be beneficial for receptor-positive subtypes but elicits unfavorable responses in triple-negative BCa (TNBC). The mechanistic basis for these conflicting effects of GC in BCa is poorly understood. In this study, we sought to decipher the molecular mechanisms that govern the GC-dependent induction of the tumor suppressor ERRFI1 gene, an inhibitor of epidermal growth factor receptor (EGFR) signaling, and characterize the role of the GC-ERRFI1 regulatory axis in TNBC. Treatment of TNBC cell lines with a protein synthesis inhibitor or GC receptor (GR) antagonist followed by gene expression analysis suggests that ERRFI1 is a direct GR target. Using in silico analysis coupled with enhancer-reporter assays, we identified a putative ERRFI1 enhancer that supports CORT-dependent transactivation. In orthogonal assays for cell proliferation, survival, migration, and apoptosis, CORT mostly facilitated an oncogenic phenotype regardless of malignancy status. Lentiviral knockdown and overexpression of ERRFI1 showed that the CORT-enhanced oncogenic phenotype is restricted by ERRFI1 in the normal breast epithelial model MCF10A and to a lesser degree in the metastatic TNBC line MDA-MB-468. Conversely, ERRFI1 conferred pro-tumorigenic effects in the highly metastatic TNBC model MDA-MB-231. Taken together, our findings suggest that the progressive loss of the GC-dependent regulation and anti-tumorigenic function of ERRFI1 influences BCa progression and may contribute to the unfavorable effects of GC therapy in TNBC.

Acquired resistance to combined BET and CDK4/6 inhibition in triple-negative breast cancer

BET inhibitors are promising therapeutic agents for the treatment of triple-negative breast cancer (TNBC), but the rapid emergence of resistance necessitates investigation of combination therapies and their effects on tumor evolution. Here, we show that palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule inhibitor, synergize with the BET inhibitor JQ1 in TNBC lines. High-complexity DNA barcoding and mathematical modeling indicate a high rate of de novo acquired resistance to these drugs relative to pre-existing resistance. We demonstrate that the combination of JQ1 and palbociclib induces cell division errors, which can increase the chance of developing aneuploidy. Characterizing acquired resistance to combination treatment at a single cell level shows heterogeneous mechanisms including activation of G1-S and senescence pathways. Our results establish a rationale for further investigation of combined BET and CDK4/6 inhibition in TNBC and suggest novel mechanisms of action for these drugs and new vulnerabilities in cells after emergence of resistance.

Response of Breast Cancer Cells to PARP Inhibitors Is Independent of BRCA Status.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have proven to be beneficial to patients with metastatic breast cancer with BRCA1/2 (BReast CAncer type 1 and type 2 genes) mutations. However, certain PARPi in pre-clinical studies have been shown to inhibit cell growth and promote the death of breast cancer cells lacking mutations in BRCA1/2. Here, we examined the inhibitory potency of 13 different PARPi in 12 breast cancer cell lines with and without BRCA-mutations using cell viability assays. The results showed that 5 of the 8 triple-negative breast cancer (TNBC) cell lines were susceptible to PARPi regardless of the BRCA-status. The estrogen receptor (ER) negative/ human epidermal growth factor receptor 2 (HER2) positive (ER-/HER2+) cells, SKBR3 and JIMT1, showed high sensitivity to Talazoparib. Especially JIMT1, which is known to be resistant to trastuzumab, was responsive to Talazoparib at 0.002 µM. Niraparib, Olaparib, and Rucaparib also demonstrated effective inhibitory potency in both advanced TNBC and ER-/HER2+ cells with and without BRCA-mutations. In contrast, a BRCA-mutant TNBC line, HCC1937, was less sensitive to Talazoparib, Niraparib, Rucaparib, and not responsive to Olaparib. Other PARPi such as UPF1069, NU1025, AZD2461, and PJ34HCl also showed potent inhibitory activity in specific breast cancer cells. Our data suggest that the benefit of PARPi therapy in breast cancer is beyond the BRCA-mutations, and equally effective on metastatic TNBC and ER-/HER2+ breast cancers.

MiR-125a Induces HER2 Expression and Sensitivity to Trastuzumab in Triple-Negative Breast Cancer Lines.

The EGFR/HER2 signaling network is an effective therapeutic target for HER2-positive cancers, which are known for their aggressive biological course. Evidence indicates that the EGFR/HER2 network plays a role in the aggressive basal-like subtype as well. Here, we studied the potential role of miR-125a-3p as a modulator of the EGFR/HER2 pathway in basal-like breast cancer. Over-expression of miR-125a-3p reduced the migratory capability of MDA-MB-231 cells and led to an increase in the expression of ErbB2 transcript and protein. The induced ErbB2 responded to trastuzumab and underwent internalization and subsequent intra-lysosomal degradation. Trastuzumab treatment further reduced the migratory capability and induced the apoptosis of the cells. An in-vivo mouse model, which supported the in-vitro findings, showed a synergistic effect for miR-125a-3p and trastuzumab. Trastuzumab-treated miR-125a-3p-induced tumors were significantly smaller than control induced tumors. Our findings indicate that, in the basal-like subtype of breast cancer, miR-125a-3p may act as a tumor suppressor. miR-125a-3p induces an increase in the expression of ErbB2 that may render the cells suitable for treatment with anti-HER2 therapies.

34P - Verification of metabolic regulatory mechanisms in androgen receptor-positive triple negative breast cancer

BackgroundAndrogen receptor-positive breast cancer, characterized by overexpression of androgen receptors (ARs), is attracting attention as a subcategory of triple-negative breast cancer (TNBC). The involvement of androgen signaling and the presence of endocrine activity in these TNBC cells make them potential treatment targets. Recent improvements in metabolomics measurement techniques have also revealed the metabolic characteristics of cancer cells. Different cancer cell subtypes exhibit different metabolic balances, and it is believed that a shift to aerobic glycolysis occurs in TNBC, although oxidative phosphorylation has been heavily implicated in endocrine-dependent breast cancer. In this study, we used metabolomics to investigate metabolic regulatory mechanisms in androgen receptor-positive TNBC. MethodsWe introduced ARs into the MDA-MB-231 strain of TNBC cells by using the pEGFP-C1-AR plasmid vector, establishing a stable AR-forced expression TNBC line (MDA-MB-231-AR). Positively ionized metabolites were purified from the parental cell line and the AR forced expression cell line. Metabolome analysis was conducted using a capillary electrophoresis–mass spectrometer (CE-TOFMS/-QqQMS). Data analysis was carried out by extracting, cross-checking, and ordering peaks using MasterHands. ResultsHierarchical clustering analysis of metabolite peak values showed that the MDA-MB-231-AR strain had improved biological malignancy compared to the parental strain. An evaluation of energy metabolism pathways (the glycolysis system, the pentose phosphate pathway, and tricarboxylic acid cycle) showed that the MDA-MB-231-AR strain had a higher NADH/NAD+ ratio and a lower lactic acid/pyruvic acid ratio than the parental strain, suggesting improved hypoxic metabolism. The glutathione/oxidized glutathione ratio was also lower in the MDA-MB-231-AR cell line than in the parental strain, indicating release from oxidative stress. ConclusionsImproved hypoxic metabolism was observed in the energy metabolism pathways of AR-positive TNBC cells. This suggests a possible shift from aerobic glycolysis to oxidative phosphorylation might in TNBC cells. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.