Breast Cancer Cell Line Models Research Articles

ADGRF1 promotes tumorigenesis via the activation of STAT3 pathway in HER2+ and triple‐negative breast cancers

Adhesion G protein‐coupled receptors (aGPCR) regulate many cellular processes involved in cancer. ADGRF1, a class VI aGPCR, is overexpressed and/or predicts poor survival in many cancer types. We previously reported that in breast cancer (BC), ADGRF1 is highly expressed in human epidermal growth factor receptor‐2 positive (HER2+) and basal‐like subtype, 85% of which are triple‐negative. Also, higher ADGRF1 expression predicts worse BC‐specific survival in both HER2+ and triple‐negative BCs patients. We previously showed that inducible ADGRF1 overexpression promoted and knockdown inhibited tumorigenesis in HER2+ BC cells. Consistently, ADGRF1 overexpression promoted tumor growth in vivo. We also reported that ADGRF1 coupling to Gαs was responsible for its pro‐tumorigenic effects. However, the downstream mediators of tumorigenesis promoted by ADGRF1 signaling remain largely unknown in cancer. Since STAT3 is a well‐known mediator of tumorigenesis in many cancers, and previous studies have shown that ADGRF1 modulates STAT3 activation in some cancers, we aimed to evaluate the effects of genetic and pharmacologic modulation of ADGRF1 on STAT3 phosphorylation (Y705) in HER2+ and triple‐negative breast cancers models. In a HER2+ BC cell line model (BT474), we found that ADGRF1 overexpression increased STAT3 phosphorylation (Y705) in vitro and in vivo. ADGRF1 knockdown also decreased STAT3 phosphorylation in BT474 cells and MDA‐MB‐231 cells, a triple‐negative BC cell line with high endogenous ADGRF1 gene expression and amplification. ADGRF1 knockdown inhibited cell growth as well as mammosphere and colony formation (a measure of tumorigenesis) in triple‐negative BC cell lines. Synaptamide increased secondary mammosphere formation, STAT3 phosphorylation (Y705), and cAMP but not IP1 in MDA‐MB‐231 cells while it had no effect on BT474 cells with inducible ADGRF1 overexpression. Synaptamide‐induced STAT3 phosphorylation was reversed by SQ22536, an adenylyl cyclase inhibitor. Our data suggest that ADGRF1 promotes tumorigenesis by coupling to Gas, which in turn activates STAT3 in HER2+ and triple‐negative BCs.

CREBBP/EP300 acetyltransferase inhibition disrupts FOXA1-bound enhancers to inhibit the proliferation of ER+ breast cancer cells.

Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer.

Abstract P1-18-17: Phase I study of cfi-402257, an oral ttk inhibitor, in patients with advanced solid tumors with breast cancer expansion cohorts

Abstract Background: TTK (also known as Mps1), a dual-specificity serine-threonine kinase, is critical for the spindle assembly checkpoint (SAC), chromosome alignment and error correction in mitosis. Inhibition of TTK causes premature mitotic exit with unattached chromosomes, resulting in chromosomal missegregation, aneuploidy and cell death. CFI-402257 is a potent (Ki = 0.09 nM, IC50 = 1.2 nM), highly selective and orally active inhibitor of TTK. Robust suppression of tumor growth was achieved upon oral dosing of single agent CFI-402257 in ER+/HER2- and triple negative breast cancer (TNBC) cell line and patient derived xenograft models. CFI-402257 demonstrated enhanced cytotoxicity in CDK4/6 inhibitor resistant ER+ breast cancer cell line models compared to parental cell lines, including those with RB1 loss. Methods: This multi-center Phase I dose escalation study (3+3 design) was designed to determine the safety, tolerability and maximum tolerated dose (MTD) of CFI-402257 and evaluate anti-tumor activity at the RP2D. CFI-402257 was dosed once daily on a continuous schedule in 28-day cycles at a starting dose of 5mg based on preclinical toxicology. Dose escalation included patients with advanced solid tumors and dose expansion at the RP2D into three expansion cohorts - Cohort A advanced solid tumors, Cohort B advanced ER+ or TNBC with 1-4 prior lines of chemotherapy for metastatic disease and Cohort C ER+/HER2- breast cancer in combination with Fulvestrant (500mg IM Day 1, 15 and 29 and then every 28 days) who have had prior treatment with an aromatase inhibitor in combination with a CDK4/6 inhibitor (>= 3 months) and =<1 prior chemotherapy for metastatic disease. Results: As of May 25, 2021, 66 patients had been enrolled, majority (76%) of patients received >3 prior therapies before study entry and 37% of patients’ (23/66) primary malignancy was breast cancer. The maximum administered dose was 294 mg and the study has continued enrolling at the recommended phase 2 dose of 168 mg with grade 3 Neutropenia, grade 3 Febrile Neutropenia and grade 3 Colitis as the dose limiting toxicities. Only 1/6 patients experienced a DLT at 168 mg (grade 3 Neutropenia, >7 days) The most common treatment emergent AEs (Gd3, >5%) were Neutropenia (15.6%), Anaemia (7.8%), Hypophosphataemia and Febrile Neutropenia (6.3%). PR’s confirmed by the Investigator have been seen to date in 33% of the cohort C subjects (2/6 patients), with an additional two cPR’s seen in the breast cancer population (ER+/HER2-) from the expansion cohorts. Conclusion: CFI-402257 is generally well tolerated and continues to enroll at 168mg daily with a manageable AE profile and early signs of anti-tumor activity. Enrollment in the expansion cohorts is ongoing and updated safety and efficacy data for the previously treated ER+/HER2- population will be presented at the time of the meeting. A multi-center phase II clinical trial is planned. Citation Format: John Hilton, Daniel J Renouf, David W Cescon, Aaron R Hansen, Albiruni RA Razak, Lee-Anne Stayner, Trisha Denny, Graham Fletcher, Tak W Mak, Mark Bray, Philippe L Bedard. Phase I study of cfi-402257, an oral ttk inhibitor, in patients with advanced solid tumors with breast cancer expansion cohorts [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 P1-18-17.

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model.

Pteridines include folate-derived metabolites that have been putatively associated with certain cancers in clinical studies. However, their biological significance in cancer metabolism and role in cancer development and progression remains poorly understood. The purpose of this study was to examine the effects of tumorigenicity on pteridine metabolism by studying a panel of 15 pteridine derivatives using a progressive breast cancer cell line model with and without folic acid dosing. The MCF10A progressive breast cancer model, including sequentially derived MCF10A (benign), MCF10AT (premalignant), and MCF10CA1a (malignant) cell lines were dosed with 0, 100, and 250 mg/L folic acid. Pteridines were analyzed in both intracellular and extracellular contexts using an improved high-performance liquid chromatography-tandem mass spectrometry method. Pteridines were located predominately in the extracellular media. Folic acid dosing increased extracellular levels of pterin, 6-hydroxylumazine, xanthopterin, 6-hydroxymethylpterin, and 6-carboxypterin in a dose-dependent manner. In particular, pterin and 6-hydroxylumazine levels were positively correlated with tumorigenicity upon folate dosing. Folic acid is a primary driver for pteridine metabolism in human breast cell. Higher folate levels contribute to increased formation and excretion of pteridine derivatives to the extracellular media. In breast cancer, this metabolic pathway becomes dysregulated, resulting in the excretion of certain pteridine derivatives and providing in vitro evidence for the observation of elevated pteridines in the urine of breast cancer patients. Finally, this study reports a novel use of the MCF10A progressive breast cancer model for metabolomics applications that may readily be applied to other metabolites of interest.

Dynamic Monitoring of EMT in CTCs as an Indicator of Cancer Metastasis.

Epithelial to mesenchymal transition (EMT) results in the genesis of circulating tumor cells (CTCs) from tumor sites and promotes the metastatic capability of CTCs in circulation. In this study, we develop a multiplex surface-enhanced Raman scattering nanotechnology for comprehensive characterization of EMT-associated phenotypes in CTCs, to monitor cancer metastasis. We observe the downregulation of the CTC marker (EpCAM) and the epithelial marker (E-cadherin), as well as the upregulation of a mesenchymal marker (N-cadherin) and a stem cell marker (ABCB5) during the transforming growth factor-β-induced EMT process in breast cancer cell line models. Additionally, we also find changes in the heterogeneity levels of these selected markers in cells. With this method, we successfully detect the presence of disease in samples from breast cancer patients and characterize EMT-associated phenotypes in their CTCs. Overall, this approach and findings provide a new means for monitoring the EMT process in cancer, insights into the detailed mechanistic progress of the diseases, and have potential for detecting the early occurrence of cancer metastasis.

Breast cancer dormancy is associated with a 4NG1 state and not senescence

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

Identification of miRNAs and related hub genes associated with the triple negative breast cancer using integrated bioinformatics analysis and in vitro approach

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype generally associated with younger women. Due to the lack of suitable drugable targets in TNBC, the microRNAs are considered as a better hope as therapeutic agents for the management of the disease. In this study, we identified differentially expressed miRNAs (DEMs) and associated hub genes in TNBC microarray data (GSE38167, GSE60714, and GSE10833) using bioinformatics tools. The identified miRNAs and genes were validated in the TNBC cell line model (MDA-MB-231) compared with the normal breast cells (MCF-10A) using the qRT-PCR technique. False-positive DEMs were avoided by comparing the DEMs profile of TNBC and triple positive breast cancer (TPBC) cell line model (BT474) compared with the MCF-10A cells data. In addition, we studied the effect of anticancer phytochemicals on the differential expression of miRNAs and genes in MDA-MB-231 cells. Furthermore, target predictions, functional enrichment and KEGG pathway analysis, mutation and copy number alterations, and overall survival analysis of DEMs in TNBC sample was investigated using standard computational tools. The study identifies first time the association of hsa-miR-1250, has-miR-1273, and has-miR-635 with the TNBC. DEMs showed significant association with the Wnt, ErbB, PI3-Akt and cAMP signaling pathways having clinical implications in TNBC tumorigenesis. The DEMs and hub genes (HOXC6 and ACVR2B) showed survival disadvantages in TNBC patients. In summary, the identified miRNAs and hub genes show important implications in TNBC tumorigenesis and patient survival. We recommend further experimental studies on pathophysiological mechanism of the identified miRNAs and hub genes in TNBC. Communicated by Ramaswamy H. Sarma

Abstract 1130: First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation

Abstract KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [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 1130.

Abstract 589: CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer

Abstract Background: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and endocytosis may identify highly aggressive breast cancer (BC) subtypes likely to spread via the circulatory system. We first studied the activation and endocytosis of CCR5 in response to its ligand CCL5 (RANTES) in the model BC cell line MDA-MB231. We then screened two types of circulating tumor-associated cells (TACs) with known negative clinical outcomes, 1) circulating tumor cells (CTCs) and 2) cancer-associated macrophage-like (CAMLs) cells, to evaluate CCR5 upregulation in relation to disease progression in metastatic breast cancer (mBC). Methods: MB231 cells were used to visualize CCR5 activation within the cell's surface and intracellular space using an anti-CCR5 antibody after stimulation with RANTES. MB231 cells were stained with LAMP-1A to verify co-localization of CCR5 and RANTES to endosomes. Anonymized peripheral blood samples from (n=54) mBC patients were obtained in accordance with local IRB regulations and with informed consent. TACs were isolated using a low-flow CellSieve Microfiltration system and stained for Cytokeratin, CD45, and CCR5. CCR5 expression signal and localization were evaluated on all TACs via fluorescence microscopy. Results: CCR5 in MB231 cells appeared as ~1μm clusters known as “CCR5 pools”, which can be found on the cell's surface, within the cytoplasm, and in the nuclear space. CCR5 pools were upregulated in the presence of RANTES, which appears to relate to endocytosis and nuclear translocation. Within the TACs of mBCs, we found similar patterns of CCR5 pooling with 70% of patients (n=38) having CCR5+ CAMLs and 41% (n=22) having CCR5+ CTCs. It was found that higher numbers of CCR5 pools (≥10pools/cell) were correlated to a 2-fold increase in both CTCs and CAMLs in circulation. In the analysis of outcome data, it was determined that patients having ≥10 CCR5 pools on their CAMLs was a highly significant predictor of worse progression free survival (PFS) and overall survival (OS) (PFS HR=4.8, CI 95% 1.9-11.6, p=0.0015) and (OS HR=3.6, CI 95% 1.3-10.0, p=0.0331). In CTCs ≥10 pools/cell was not a significant predictor for worse PFS and OS. However, ≥5 CCR5 pools/cell significantly predicted for worse PFS but not OS (PFS HR=2.9, CI 95% 1.2-7.4, p=0.0418 and OS HR=3.3, CI 95% 1.1-9.3, p= 0.0539). Conclusion: We observed the activation and endocytosis of CCR5 pools using RANTES in MD-MB231 cell lines, and then demonstrated that this upregulated motility pathway appears to be conserved in migratory cells in mBC patients. Further, this upregulation of CCR5 appears to correlate with worse clinical outcomes, which may provide an explanation for how tumor-associated cells move into circulation and acquire metastatic potential. Citation Format: Ashvathi Raghavakaimal, Massimo Cristofanilli, Cha-Mei Tang, R. K. Alpaugh, Kirby P. Gardener, Saranya Chumsri, Daniel L. Adams. CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer [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 589.

Differential Genomic Interactions Drive Progesterone Receptor Isoform Specific Functions in Breast Cancer

Progesterone is critical for normal breast development and function, and has been shown to stimulate proliferation of normal breast epithelial cells by increasing stem and progenitor cell numbers. Breast cancer incidence is increased in women exposed to progesterone analogues in combined estrogen plus progestin hormone replacement therapy, but not in women taking estrogen alone. Classical progesterone signaling is mediated through the nuclear progesterone receptor (PR), which occurs as two related but functionally different isoforms, PRA and PRB. PRA and PRB are co-expressed equally in normal breast tissue but become dysregulated in breast cancer where PRA often becomes predominant. PRA predominance in breast cancer is associated with poorer outcome and higher risk of distant metastasis in tamoxifen treated patients. We show using integrated analysis of ChIP-seq, ATAC-seq and transcriptomic profiling in a breast cancer cell line model of acquired PRA predominance that: 1) PRA and PRB have different requirements with regard to chromatin accessibility; 2) PRA predominance reshapes the PR cistrome and the associated transcriptome to affect genes not normally regulated by PR when PRA and PRB are equivalently expressed, possibly through assisted loading with multiple other transcription factors; 3) Genes regulated by PR only when PRA is predominant are associated with poorer breast cancer outcome and involved in multiple cancer-associated pathways including those that regulate cell proliferation and adhesion. Our data suggest a mechanism for the poorer disease outcome seen in breast cancers with a predominance of PRA.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2‐positive breast cancer

Purpose Adhesion G protein coupled receptors (aGPCRs) remains least explored GPCRs family for their role in health and disease. The aGPCR, ADGRF1 (GPR110), is overexpressed and predicts poor survival in various cancer types. However, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We have reported that ADGRF1 is overexpressed in tumorigenic population and in anti-HER2 therapy-resistant derivatives of various HER2+ breast cancer (BC) cell line models. Further, we have reported that ADGRF1 knockdown reduces tumorigenesis and metastasis in HER2+ BC cells. In this study, we evaluated the effects of ADGRF1 overexpression (OE) on tumorigenesis, signaling pathways, and outcomes in HER2+ BC. Methods Stable clones of 2 HER2+ BC cell lines, BT474 and SKBR3, were generated to overexpress ADGRF1 construct in doxycycline-inducible manner. The effect of ADGRF1 on tumorigenesis was evaluated in vitro using soft agar assays, mammosphere assay, and Aldefluor assay. The effect on the HER pathway was evaluated by assessing expression of phospho- and total-HER1 and -HER2 and potency of anti-HER2 drugs. G protein coupling of ADGRF1 was determined by co-immunoprecipitation of Gαs and Gαq and detecting second messengers, cAMP and IP1, in presence/absence of synaptamide, an ADGRF1 agonist. Downstream pathways of ADGRF1 were explored by RNAseq and Reverse Phase Protein Arrays (RPPA) and functional assays. ADGRF1 expression was assessed in BC patients from The Cancer Genome Atlas. ADGRF1 OE effects on survival was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium dataset. Results ADGRF1 OE enhanced the number of colonies and mammospheres and increased Aldefluor+ tumorigenic cell population, suggesting a role in tumorigenesis. ADGRF1 OE did not alter the expression total- or phospho-HER1/-HER2 proteins or efficacy of anti-HER2 drugs. Overexpressed ADGRF1 co-immunoprecipitated with Gαs and Gαq proteins and increased cAMP and IP1. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 OE, suggesting pro-tumorigenic effects of the ADGRF1-Gαs pathway. Synaptamide, did not alter cAMP or IP1 with/without ADGRF1 OE or the number of mammospheres. Integration of RNA-Seq and RPPA data showed that ADGRF1 OE inhibited cell cycle. We detected G0/1 arrest by cell cycle analysis with ADGRF1 OE. The protein level of the proliferation marker Ki67 was reduced upon ADGRF1 OE. Also, ADGRF1 OE led to a 10-fold reduction in potency of docetaxel, a chemotherapy drug used in HER2+ BC. ADGRF1 was overexpressed and amplified in basal and HER2 compared to luminal A and B BCs. High ADGRF1 expression predicted poor survival in HER2 subtype, but not in basal BC. Conclusions: ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

Innovative Betulin Nanosuspension exhibits enhanced anticancer activity in a Triple Negative Breast Cancer Cell line and Zebrafish angiogenesis model

We present a nanosuspension of betulin, a BCS class II anticancer drug, particularly effective against resistant breast cancer. As anticancer efficacy of betulin is hampered by poor aqueous solubility, a nanosuspension with surface area was considered to enhance efficacy. An innovative approach wherein the betulin nanosuspension is generated instantaneously in situ, by adding a betulin preconcentrate (BeTPC) comprising drug and excipients, to aqueous medium, is successfully demonstrated. The optimal BeTPC when added to isotonic dextrose solution instantaneously generated an in situ nanosuspension (BeTNS-15) with high precipitation efficiency (92.7 ± 1.21%), average particle size (383.74 ± 7.24 nm) and good stability as per ICH guidelines. TEM revealed elongated particles while DSC and XRD indicated partial amorphization. Significantly higher cytotoxicity of BeTNS-15 (IC50 38.44 µg/ml) compared to betulin (BetS) (IC50 69.54 µg/ml) in the resistant triple negative human breast cancer cell line MDA-MB-231, was attributed to high intracellular uptake confirmed by HPLC and Imaging Flow cytometry (IFC). IFC confirmed superior anti-cancer efficacy of BeTNS-15 mediated by mitochondrial membrane disruption and inhibition of the G0/G1 phase. BeTNS-15 also exhibited significantly greater anti-angiogenic efficacy (p < 0.05) in the zebrafish model confirming superior efficacy. Simplicity of the innovative in situ approach coupled with superior efficacy proposes BeTNS as an innovative and highly promising anticancer formulation.

Identifying Molecular Signatures of Distinct Modes of Collective Migration in Response to the Microenvironment Using Three-Dimensional Breast Cancer Models

Simple SummaryThe objective of this study was to investigate the role of two microenvironmental factors, namely, tumor-intrinsic hypoxia and secretome in inducing collective migration. We utilized three-dimensional (3D) discrete-sized microtumor models, which recapitulate hallmarks of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Tumor-intrinsic hypoxia induced directional migration in large hypoxic microtumors while secretome from large microtumors induced radial migration in non-hypoxic microtumors. This highlights the emergence phenotypic heterogeneity and plasticity in cancer cells in response to different microenvironmental stimuli. To unravel mechanisms underlying these two distinct modes of migration, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory phenotypes using non-migratory, non-hypoxic microtumors as controls. We proposed unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration.Collective cell migration is a key feature of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) among many other cancers, yet the microenvironmental factors and underlying mechanisms that trigger collective migration remain poorly understood. Here, we investigated two microenvironmental factors, tumor-intrinsic hypoxia and tumor-secreted factors (secretome), as triggers of collective migration using three-dimensional (3D) discrete-sized microtumor models that recapitulate hallmarks of DCIS-IDC transition. Interestingly, the two factors induced two distinct modes of collective migration: directional and radial migration in the 3D microtumors generated from the same breast cancer cell line model, T47D. Without external stimulus, large (600 µm) T47D microtumors exhibited tumor-intrinsic hypoxia and directional migration, while small (150 µm), non-hypoxic microtumors exhibited radial migration only when exposed to the secretome of large microtumors. To investigate the mechanisms underlying hypoxia- and secretome-induced directional vs. radial migration modes, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory microtumors compared with non-hypoxic, non-migratory small microtumors as controls. We propose unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration. Gene Set Enrichment Analysis (GSEA) and protein-protein interaction (PPI) network analysis revealed enrichment and potential interaction between hypoxia, EMT, and migration gene signatures for the hypoxia-induced directional migration. In contrast, hypoxia and EMT were not enriched in the secretome-induced radial migration, suggesting that complete EMT may not be required for radial migration. Survival analysis identified unique genes associated with low survival rate and poor prognosis in TCGA-breast invasive carcinoma dataset from our tumor-intrinsic hypoxia gene signature (CXCR4, FOXO3, LDH, NDRG1), hypoxia-induced EMT gene signature (EFEMP2, MGP), and directional migration gene signature (MAP3K3, PI3K3R3). NOS3 was common between hypoxia and migration gene signature. Survival analysis from secretome-induced radial migration identified ATM, KCNMA1 (hypoxia gene signature), and KLF4, IFITM1, EFNA1, TGFBR1 (migration gene signature) to be associated with poor survival rate. In conclusion, our unique 3D cultures with controlled microenvironments respond to different microenvironmental factors, tumor-intrinsic hypoxia, and secretome by adopting distinct collective migration modes and their gene expression analysis highlights the phenotypic heterogeneity and plasticity of epithelial cancer cells.

A novel naphthalimide that selectively targets breast cancer via the arylhydrocarbon receptor pathway

We report that the naphthalimide analogue 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6) is a highly potent and selective breast cancer targeting molecule. These effects are mediated via the aryl hydrocarbon receptor (AHR) pathway and the subsequent induction of CYP1 metabolising monooxygenases in breast cancer cell line models. Indeed the triple negative breast cancer cell line MDA-MB-468 with a GI50 value of 100 nM is greater than 500-fold more sensitive to NAP-6 compared with other tumour derived cell models. Within 1 h exposure of these cells to NAP-6, CYP1A1 expression increases 25-fold, rising to 250-fold by 24 h. A smaller concurrent increase in CYP1A2 and CYP1B1 is also observed. Within 24 h these cells present with DNA damage as evident by enhanced H2AXγ expression, cell cycle checkpoint activation via increased CHK2 expression, S-phase cell cycle arrest and cell death. Specific small molecule inhibitors of the AHR and CYP1 family ameliorate these events. A positive luciferase reporter assay for NAP-6 induced XRE binding further confirms the role of the AHR in this phenomenon. Non-sensitive cell lines fail to show these biological effects. For the first time we identify 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione as a new AHR ligand that selectively targets breast cancer.

Abstract 4689: Co-treatment of MEKi and BRD4 inhibitors lead to synergistic repression of MYC-associated enhancer RNAs

Abstract Recent evidence suggests that co-treatment with BRD4 and MEK inhibitors can lead to synergistic suppression of MAPK signaling, however, little is known about the mechanism of action of this synergy. In a triple negative breast cancer cell line model, we harvested nascent RNA at 45 minutes following repression of BRD4 and MEK via a variant of the precision run-on followed by sequencing (PRO-seq) assay. We noted dose-dependent repression of enhancer RNAs over MYC binding sites as well as enhancer RNAs upstream known ERK targets such as DUSP6. Using new machine learning techniques, we were able to link synergy-specific enhancer RNAs to their target genes. This work will inform on new methods for patient stratification and biomarker selection of the BETi and MEKi treatment regimes. Citation Format: Joseph Azofeifa, Joel Basken, Maria Lai, Ryan Langendorf, Laura Norris, Tim Read, Adam Robbins-Pianka. Co-treatment of MEKi and BRD4 inhibitors lead to synergistic repression of MYC-associated enhancer RNAs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4689.

Abstract 520: Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines

Abstract Parathyroid Hormone-related Peptide (PTHrP)is a potent regulator of bone turnover and is thought to play a major role in the progression of skeletal metastasis through its activation of growth factors in the bone microenvironment. Our previous studies using genetically engineered mouse models in which PTHrP was ablated in the mammary epithelium have demonstrated that PTHrP also plays a direct role on tumor progression by enhancing tumor initiation, growth and metastasis outside the skeleton. We therefore proposed a novel mechanism of PTHrP driven bone metastasis by which PTHrP controls both the seed (tumor cells) and the soil (the bone microenvironment). We tested our hypothesis in triple negative breast cancer cell line (TNBC)models which are not responsive to either tamoxifen or Herceptin. Monoclonal antibodies against PTHrP were generated with strong in vitro anti-proliferative and anti-invasive potency in two human PTHrP expressing cell lines: the widely used TNBC MDAMB231 cell line and a patient derived TNBC cell line. Ablation of PTHrP or treatment with anti-PTHrP monoclonal antibodies induced major changes in the phenotype of these cells as determined by flow cytometry with stem cells and EMT markers, invasion assays and mammosphere assays indicating a reversal of their metastatic/invasive phenotype. Next we transplanted these cell lines into 8 weeks old female athymic nude mice to examine the efficacy of the therapeutic monoclonal antibodies on the progression of established bone metastasis.104 tumor cells were injected intra-tibially and animals examined at timed intervals(14, 21, 28, 25 and 42 days) with X-rays and high resolution CTs.At 14 days animals were administered intra-peritoneally with 200 micrograms of therapeutic anti-PTHrP antibodies or non-immune IgG(10 animals per group) and every 3 days thereafter til sacrifice(42 days). At sacrifice PET scan was performed to measure tumor volume and activity and bone were collected for histomorphometry analysis of tumor size and bone turnover activity. A significant reduction in bone lesions (P&amp;lt;0.05) was observed over time by imaging in animals treated with the therapeutic antibodies. At sacrifice PET scan indicates a strong reduction in tumor activity in treated animals (P&amp;lt;0.05). Histomorphometry analysis showed greater than 50% reduction in tumor volume associated with a significant inhibition of bone turnover in treated animals (P&amp;lt;0.05). In summary our data demonstrate that, in two TNBC models, PTHrP blockade can reprogram cancer cells to a reduced tumorigenicity and block the progression of established bone metastasis by targeting both the tumor cell and the bone microenvironment. Our data lend support to the “seed and soil” hypothesis proposed by Paget and the rational of anti-PTHrP therapeutic strategies alone or in combination with bone targeting agents in order to eradicate the development of skeletal metastasis. Citation Format: Jiarong Li, Anne Camirand, Mahvash Zakikhani, Karine Sellin, Richard Kremer. Therapeutic antibodies against parathyroid hormone-related peptide (PTHrP) inhibit the growth of established skeletal metastasis in mice transplanted with human triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 520.

Reciprocal fine-tuning of progesterone and prolactin-regulated gene expression in breast cancer cells

Progesterone and prolactin are two key hormones involved in development and remodeling of the mammary gland. As such, both hormones have been linked to breast cancer. Despite the overlap between biological processes ascribed to these two hormones, little is known about how co-expression of both hormones affects their individual actions. Progesterone and prolactin exert many of their effects on the mammary gland through activation of gene expression, either directly (progesterone, binding to the progesterone receptor [PR]) or indirectly (multiple transcription factors being activated downstream of prolactin, most notably STAT5). Using RNA-seq in T47D breast cancer cells, we characterized the gene expression programs regulated by progestin and prolactin, either alone or in combination. We found significant crosstalk and fine-tuning between the transcriptional programs executed by each hormone independently and in combination. We divided and characterized the transcriptional programs into four broad categories. All crosstalk/fine-tuning shown to be modulated by progesterone was dependent upon the expression of PR. Moreover, PR was recruited to enhancer regions of all regulated genes. Interestingly, despite the canonical role for STAT5 in transducing prolactin-signaling in the normal and lactating mammary gland, very few of the prolactin-regulated transcriptional programs fine-tuned by progesterone in this breast cancer cell line model system were in fact dependent upon STAT5. Cumulatively, these data suggest that the interplay of progesterone and prolactin in breast cancer impacts gene expression in a more complex and nuanced manner than previously thought, and likely through different transcriptional regulators than those observed in the normal mammary gland. Studying gene regulation when both hormones are present is most clinically relevant, particularly in the context of breast cancer.

Abstract P5-05-01: Novel mechanisms of action of selective androgen receptor modulator RAD140 in AR+/ER+ breast cancer models

Abstract Background: Recurrence after standard-of-care estrogen receptor α (ER)-targeted therapy presents challenges in management of metastatic ER positive (ER+) breast cancer (BC). The high prevalence of androgen receptor (AR) in ER+ BC, along with preclinical and clinical evidence showing anti-tumor activity of AR agonists in AR+/ER+ BC, suggest an opportunity to address resistance to ER-targeted agents by targeting AR. Significant anti-tumor activity has been reported with RAD140, an oral selective AR modulator (SARM), in estrogen-dependent and -independent AR+/ER+ BC models. Mechanistic investigation revealed that inhibition of tumor growth by RAD140 was in part through AR-mediated reduction of ESR1 mRNA and subsequent suppression of ER signaling, independent of ESR1 alterations. Here, we present new observations on RAD140 mechanisms of action in in vitro and in vivo models of endocrine-independent BC models. Methods: HCC1428-long term estrogen deprived (LTED) line was established by passaging cells in steroid-depleted media for &amp;gt; 6 months. HCC1428-LTED-palbociclib resistant (-palboR) BC cell line was generated from LTED by long-term passaging in the presence of 500 nM palbociclib. Fluorescence-microscopy was used to assess annexin V/propidium iodide (PI) and phospho- H2A.X expression. The endocrine-independent BC PDX model was established in nude mice without supplemental estradiol. Prior anti-cancer therapy of the donor patient included tamoxifen, exemestane, fulvestrant and multiple lines of chemotherapy. RAD140 (3 mg/kg twice daily), elacestrant (30 mg/kg once daily), and palbociclib (10 mg/kg once daily) were administered via oral gavage for the duration of study. Fulvestrant (3 mg/mouse) was given once weekly subcutaneously. Results: In a hormone-independent BC PDX model harboring the ESR1 D538G mutation, RAD140 treatment led to 80% tumor growth inhibition (TGI) compared to the vehicle. The selective ER degrader (SERD) fulvestrant had TGI of 7%, while the oral SERD elacestrant (RAD1901) had TGI of 60%. The combination of RAD140 with elacestrant exhibited enhanced anti-tumor activity greater than that of each agent alone (93% TGI). These in vivo findings suggest the activity of the AR-targeting RAD140 may be mediated through mechanisms beyond its AR-mediated inhibition of ER signaling. We then sought to identify additional mechanisms of action of RAD140 in two hormone-independent AR+/ER+ BC cell line models, HCC1428-LTED and HCC1428-LTED-palboR. After prolonged treatment (2 weeks) at 100 nM, RAD140 induced morphological changes related to apoptosis, along with increased number of annexin V/PI-positive cells. This was further supported by increases in cleaved PARP/caspase3/caspase7 proteins by western blot analysis. A decrease in the pro-survival protein BCL-2 was also seen. These results collectively suggest that RAD140 induced apoptosis in endocrine-resistant BC cells. In line with these findings, an increase in phospho-H2A.X, a sensitive marker for DNA damage, was also observed in RAD140-treated cells. Similar changes were observed with the positive control AR agonist dihydrotestosterone (DHT), but not with fulvestrant. The AR antagonist apalutamide was found to partially reverse the apoptosis induced by RAD140 and DHT, further supporting the on-target effect. Conclusion: Our data suggest the anti-tumor activity of RAD140 may be mediated through suppression of ER signaling as well as additional mechanisms. The effect of RAD140 on DNA damage and apoptosis implies the potential for combination therapy with agents targeting cell survival and DNA damage repair pathways. RAD140 is being studied in a Phase 1, first-in-human trial in postmenopausal women with hormone receptor positive BC (NCT03088527). Citation Format: Suqin He, Maureen G Conlan, Heike Arlt, Charles Morris, Ziyang Yu. Novel mechanisms of action of selective androgen receptor modulator RAD140 in AR+/ER+ breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-01.

Abstract P6-03-20: The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance

Abstract Introduction: Neratinib is an irreversible, small molecule pan-HER tyrosine kinase inhibitor that has shown clinical activity against HER2-amplified (HER2+) and HER2-mutated breast cancer. However, there are limited data on potential mechanisms of resistance to neratinib. This study aimed to develop HER2+ breast cancer cell line models of acquired neratinib resistance, and to elucidate the effects of acquired resistance on HER family signalling and drug response. As neratinib has activity in both estrogen receptor positive (ER+) and ER- HER2+ breast cancer, neratinib-resistant cell lines were generated and characterised from breast cancer cell lines of both subtypes. Methods: HER2+, ER- HCC1569 cells were continuously exposed to 150 nM neratinib for six months to generate the neratinib-resistant HCC1569 (HCC1569-N) cell line. HER2+ ER+ BT474 cells were continuously exposed to escalating doses of 5-80 nM neratinib for six months to generate the BT474-N cell line. Sensitivity to HER2-targeted therapies (neratinib, lapatinib and trastuzumab) and chemotherapeutics (5’dFUR, carboplatin, and vinorelbine) were assessed by 5-day acid phosphatase based proliferation assay. IC50 and combination index (CI) values were calculated using CalcuSyn software. Total and phosphorylated levels of HER2, EGFR, ERK, and Src were assessed by Western blotting following neratinib treatment (150 nM), to determine changes in HER2 signalling. Results: The HCC1569-N and BT474-N cells had a 50.3-fold and 105-fold increase in neratinib resistance. In addition, both cell lines developed cross-resistance to lapatinib and were resistant at clinically relevant concentrations (&amp;gt; 1 µM). HCC1569-Par cells are innately resistant to trastuzumab, which was not altered in HCC1569-N cells. However, neratinib resistance also caused resistance to trastuzumab in the BT474-N cell line, compared to its trastuzumab-sensitive parental cell line (Table 1). The HCC1569-N cell line did not show any significant difference in sensitivity to 5’dFUR, carboplatin, or vinorelbine compared to parental HCC1569, but the addition of vinorelbine to neratinib was synergistic (CI value = 0.28 ± 0.19). HCC1569-N cells displayed no changes in total protein or phosphorylated levels of HER2 or EGFR compared to parental cells. Additionally, neratinib treatment still resulted in inhibition of HER2, EGFR, and downstream ERK signalling, indicating a lack of reliance on HER2 signalling in HCC1569-N cells. Phosphorylated Src levels were elevated (p = 6.3 × 10−4) in HCC1569-N compared to parental cells. Conclusions: Neratinib resistance in HCC1569-N and BT474-N cells confers cross-resistance to other HER2-targeted therapies. Increased Src activity may be implicated in the neratinib-resistant HER2+ ER- phenotype. Cell line10 µg/mL Trastuzumab (%Growth)NeratinibIC50 valueLapatinibIC50 valueHCC1569-Par97.6 ± 2.57.8 ± 5.8653.0 ± 144.0HCC1569-N92.7 ± 2.9393.0 ± 77.1&amp;gt; 1000BT474-Par44.7 ± 1.81.2 ± 0.323.9 ± 19.6BT474-N97.8 ± 3.3126.8 ± 53.5&amp;gt; 1000 Citation Format: Neil T Conlon, Alacoque Browne, Laura Breen, Alex J Eustace, John Crown, Denis M Collins. The development and characterization of novel HER2-positive breast cancer models of acquired neratinib resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-20.

BRAF, KRAS and PIK3CA Mutation and Sensitivity to Trastuzumab in Breast Cancer Cell Line Model.

BRAF, KRAS and PIK3CA Mutation and Sensitivity to Trastuzumab in Breast Cancer Cell Line Model.