Murine Triple-negative Breast Cancer Cells Research Articles

A BPTF Inhibitor That Interferes with the Multidrug Resistance Pump to Sensitize Murine Triple-Negative Breast Cancer Cells to Chemotherapy.

Triple-negative breast cancer (TNBC) is associated with a generally poor prognosis due to its highly aggressive and metastatic nature, lack of targetable receptors, as well as the frequent development of resistance to chemotherapy. We previously reported that AU1, a small molecule developed as an inhibitor of BPTF (bromodomain PHD finger-containing transcription factor), was capable of sensitizing preclinical models of TNBC to chemotherapy in part via the promotion of autophagy. In studies reported here, we identify an additional property of this compound, specifically that sensitization is associated with the inhibition of the P-glycoprotein (P-gp) efflux pump. In silico molecular docking studies indicate that AU1 binds to active regions of the efflux pump in a manner consistent with the inhibition of the pump function. This work identifies a novel chemical structure that can influence multidrug efflux, an established mechanism of drug resistance in TNBC, that has not yet been successfully addressed by clinical efforts.

Abstract B022: Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer (BC) cases but due to its aggressive nature and the lack of targeted treatment options, represents a disproportionately higher number of BC-related deaths. Compared to other BC subtypes, TNBCs are more inflamed with M2-like tumor-associated macrophages (TAMs) and cytotoxic T-lymphocytes (CTLs) which are associated with poor and good prognosis, respectively. Clinical trials have proven that immunogenic cancers can be effectively treated through immune checkpoint blockade (ICB), a treatment strategy that prevents cancer cells from silencing CTL-mediated attack. However, CTLs can be suppressed by M2-like TAMs which can limit ICB efficacy and support tumor growth. The hedgehog (HH) signaling pathway is highly activated in TNBC and higher expression of GLI1 is associated with worse overall survival. Recent pan-cancer analyses highlight a significant correlation between activated HH signaling and characteristics of immune evasion. We aim to clarify how activated HH signaling regulates resident innate and adaptive immune cells in the tumor microenvironment (TME) in TNBC. Methods: Primary K14-cre; Brca1 fl/fl ; P53 fl/fl (KBP) murine TNBC cells were transfected with CRISPR/Cas9 + sgRNA targeting GLI1. After isolating single cell clones and confirming GLI1 knockout (KO), KBP and KBP-GLI1-KO cells were orthotopically injected into the mammary fat pad of syngeneic immune-competent female mice and allowed to grow for 6-8 weeks. Tumor volume was measured once tumors were palpable, and tumors were harvested once the largest tumor reached ethical endpoint. Tumors were weighed at harvest and fluorescence activated cell sorting (FACS) was performed to determine if there was an effect on the composition of immune populations. Results: At harvest, KBP-GLI1-KO tumors were delayed in growth and significantly smaller than KBP tumors. FACS analysis showed that GLI1-KO tumors had increased CD4+ and CD8+ T cells and a reduced amount of Foxp3+ CD4+ Tregs. Furthermore, GLI1-KO tumors had significantly fewer F4/80+ CD11b+ TAMs within the TME. Amongst TAM populations, there was reduced CD206+ M2-like TAMs and an increase in CD80+ M1-like TAMs upon loss of GLI1. Similar trends were observed when KBP allografts were treated with the GLI1 inhibitor, GANT61. Immunohistochemical and RNAseq analysis are ongoing. Similar experiments with other KBP-GLI1-KO clones are currently being done. Conclusion: Here, we show that tumoral GLI1 not only promotes tumor growth, but also supports an immunosuppressive TME in TNBC. Abolishing GLI1 converted tumors to a pro-inflammatory phenotype marked by an increase in CD4+ and CD8+ T cells and a reduction in CD206+ M2-like TAMs. Future experiments are aimed at revealing the mechanisms by which GLI1 regulates immune cell recruitment to the TME and testing the effect of combination immunotherapy. Overall, these preliminary findings identify GLI1 as a potential therapeutic target to limit tumor growth and promote anti-tumor immunity. Citation Format: Aidan J Gray, Wanda Marini, Kiichi Murakami, Michael Reedijk. Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B022.

Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

Synergistic Activation of LEPR and ADRB2 Induced by Leptin Enhances ROS Generation in TNBC Cells.

Leptin interacts not only with leptin receptor (LEPR) but also engages with other receptors. While the pro-oncogenic effects of the adrenergic receptor β2 (ADRB2) are well-established, the role of leptin in activating ADRB2 in triple-negative breast cancer (TNBC) remains unclear. The pro-carcinogenic effects of LEPR were investigated using murine TNBC cell lines, 4T1 and EMT6, and a tumor-bearing mouse model. Expression levels of LEPR, NOX4, and ADRB2 in TNBC cells and tumor tissues were analyzed via Western blot and qPCR. Changes in reactive oxygen species (ROS) levels were assessed using flow cytometry and MitoSox staining, while immunofluorescence double-staining confirmed the co-localization of LEPR and ADRB2. LEPR activation promoted NOX4-derived ROS and mitochondrial ROS production, facilitating TNBC cell proliferation and migration, effects which were mitigated by the LEPR inhibitor Allo-aca. Co-expression of LEPR and ADRB2 was observed on cell membranes, and bioinformatics data revealed a positive correlation between the two receptors. Leptin activated both LEPR and ADRB2, enhancing intracellular ROS generation and promoting tumor progression, which was effectively countered by a specific ADRB2 inhibitor ICI118551. In vivo, leptin injection accelerated tumor growth and lung metastases without affecting appetite, while treatments with Allo-aca or ICI118551 mitigated these effects. This study demonstrates that leptin stimulates the growth and metastasis of TNBC through the activation of both LEPR and ADRB2, resulting in increased ROS production. These findings highlight LEPR and ADRB2 as potential biomarkers and therapeutic targets in TNBC.

Repurposing hemostatic drug desmopressin in AVPR2-expressing triple-negative breast cancer: Target expression analysis and preclinical antineoplastic effects on tumor growth and metastatic progression.

e15087 Background: Desmopressin (dDAVP) is a hemostatic drug that acts as an agonist of the arginine vasopressin receptor 2 (AVPR2) that is present in vascular and malignant cells. Preclinical data show that dDAVP triggers cytostatic mechanisms, blocking angiogenesis and reducing metastases by limiting the survival of circulating tumor cells. Triple-negative breast cancer (TNBC) is associated with poor prognosis due to limited response to therapy and frequent early metastatic recurrence. Considering the unsatisfied clinical need for novel therapeutic options in TNBC, we evaluated the antitumoral activity of dDAVP on experimental models of TNBC, alone or in addition to chemotherapy. We also assessed the expression and clinical impact of AVPR2 in breast cancer samples. Methods: Human and murine TNBC cell lines were used for in vitro experiments. Target expression was assessed by qPCR and immunohistochemistry (IHC), in experimental or clinical tumors (NCT01606072). Effects of dDAVP were evaluated by proliferation, clonogenic, 3D growth and chemotaxis assays, qPCR arrays, and confocal microscopy. Local and distant tumor progression were assessed in vivo in nude or BALB/c mice. TIMER2.0 and UCSC Xena platforms (BRCA TCGA /n = 1260) were used for bioinformatic studies. Results: dDAVP significantly reduced growth and chemotaxis of AVPR2-expressing TNBC cells. Cytostatic effects of dDAVP were associated with altered actin cytoskeleton dynamics and differential expression of angiogenesis and metastasis-related genes. Cooperative effects were observed in vitro and in vivo after combining dDAVP with taxane or alkylating therapy. Tumor-bearing mice were treated with intravenous injections of dDAVP (0.3 µg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10 mg/kg i.p.) or carmustine (25 mg/kg i.p.). Combination regimens resulted in greater survival and tumor growth and metastasis inhibition. Bioinformatic analysis of the TCGA database showed that AVPR2 had a positive prognostic impact on overall survival in breast cancer patients, especially in the basal-like molecular subtype. AVPR2 also correlated with different immune cell infiltrates such as NK and dendritic cells, and CD4+ and CD8+ T cells. Moreover, AVPR2 expression was detected by IHC in 6/18 breast cancer clinical samples using a small cohort of surgical specimens. Conclusions: AVPR2 expression is associated with improved outcomes in patients with TNBC. Agonist activation of AVPR2 using dDAVP leads to cancer cell growth inhibition in TNBC cell lines and mouse models. We propose dDAVP as a potential novel therapeutic option for treating TNBC either as monotherapy or in combination with chemotherapy. The therapeutic impact of its addition to immune checkpoint inhibitors remains to be explored.

Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

Abstract 708: Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC)

Abstract Purpose: Triple negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype with few treatment options. Radiation therapy (RT) is a mainstay therapy for the treatment of BC, but local recurrence following RT therapy is common. Consequently, decreasing local recurrence in patients with TNBC is a critical clinical need. Prior work demonstrated that AURKB is overexpressed in TNBC, and over-expression correlates with poor prognosis. Here, we examined the effects of AURKB inhibition as a novel radiosensitizing strategy in syngeneic TNBC models. Methods: Cell viability assays were used to determine the half-maximal inhibitory concentrations (IC50) of the AURKB inhibitors Barasertib-HQPA and SP-96 72 hours post treatment. Clonogenic survival assays were used to assess the radiosensitivity of TNBC murine cell lines to AURKB inhibition. In these assays, AURKB inhibitors were delivered at sub-IC50 concentrations 24 hours prior to RT, and radiation enhancement ratios (rERs) were calculated. Immunofluorescent microscopy using DAPI stain assessed micronuclei. Propidium iodide staining to assess aneuploidy was completed via flow cytometry. To assess the radiosensitizing effects of AURKB inhibition in vivo, the 4T1 syngeneic TNBC cell line was injected bilaterally into Balb/c mice and treated with Barasertib and RT. Tumor volume was recorded twice weekly throughout the study. Results: Aurora kinase B inhibitors (500-750 nM Barasertib-HQPA, 100-200 nM SP-96) delivered 24 hours prior to radiation therapy induced radiosensitization in the murine TNBC cells 4T1 (rER: 1.24-1.56) and Py8119 (rER: 1.51-1.72). Mechanistically, combined AURKB inhibition and RT significantly increased micronuclei formation in 4T1 cells 24 hours after RT compared to vehicle control (p<0.0001). Furthermore, combined AURKB inhibition and RT induced aneuploidy in murine TNBC cell lines 24 hours after radiation therapy compared to vehicle control. Combined AURKB inhibition (Barasertib 25 mg/kg, IP) and RT (8 Gy RT in 1 fraction) significantly decreased tumor volume compared to mice that had received vehicle control (777 ± 61 mm3 vs 1623 ± 126 mm3; p <0.0001). Conclusion: AURKB inhibition induces radiosensitization in syngeneic models of TNBC and leads to increased micronuclei and aneuploidy, suggesting a mechanism of sensitization. These results suggest that AURKB is a potential radiosensitizing strategy for the treatment of triple negative disease. Ongoing studies are further refining the underlying mechanisms of AURKB inhibition and RT on the antitumoral immune response. Citation Format: Kassidy M. Jungles, Zhuwen Wang, Caroline Bishop, Cydnee Wilson, Meilan Liu, Jadyn James, Michael Green, James M. Rae, Corey W. Speers, Lori J. Pierce. Targeting aurora kinase B (AURKB) as a radiosensitizing strategy in syngeneic models of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 708.

Abstract 7120: Investigating PARP1 selective inhibitor DSB1559 efficacy in BRCA1-associated triple negative breast cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) have improved outcomes of BRCA-associated breast cancer. However, toxicity can limit patient response as well as the ability to be combined with other therapeutics. Current PARPi broadly inhibit members of the PARP protein family, however only inhibition of PARP1 is needed for PARPi-mediated cell death. As inhibition of PARP2 is associated with hematological toxicity seen in PARPi patients, selectively inhibiting PARP1 may reduce PARPi toxicity and lead to better outcomes for patients. Here we characterize and test the efficacy of the PARP1 selective inhibitor (PARP1i) DSB1559 compared to Olaparib in BRCA1-associated triple negative breast cancer (TNBC). Methods: NanoBRET, PARylation, and pharmacokinetic/pharmacodynamic assays were performed to examine DSB1559 potency, PARP family selectivity, and in vivo behavior. Immunocompetent FVB/n mice bearing BRCA1-deficient TNBC (CMV-Cre;Brca1f/f;Trp53f/f) tumors were treated daily with PARPi (Olaparib) or PARP1i (DSB1559) for 60 days and followed for survival. CellTiter-Glo and qPCR assays were used to examine DSB1559 effect on cell viability and STING activation, respectively. Results: DSB1559 is a novel and potent PARP1 inhibitor which has superior selectivity for PARP1 compared to Olaparib and PARP1i AZD5305. DSB1559 has highly desirable physico-chemical and in vitro ADME properties, which translate to high oral bioavailability and moderate clearance in rodent PK studies. Additionally, DSB1559 demonstrated superior tumor residence time and tumor/plasma ratio compared to AZD5305. Compared to Olaparib, DSB1559 significantly increased STING activation and reduced cell viability in BRCA1-associated murine TNBC cells. BRCA1-associated TNBC-bearing immune-competent mice treated with DSB1559 had significantly reduced tumor growth and increased overall survival (median survival 74 days) compared to Olaparib (median survival 18 days). Analysis of changes in the tumor microenvironment are ongoing. Conclusions: Taken together, DSB1559 is a highly selective PARP1 inhibitor that demonstrates superior efficacy compared to Olaparib in a BRCA1-associated immune-competent TNBC model. Citation Format: Adam Nelson, Kelly F. Zheng, Carlos Wanderley, Daniel E. Michaud, Phillip M. Cowley, Gillian M. Campbell, Barry E. McGuinness, Alan Wise, Jennifer Guerriero. Investigating PARP1 selective inhibitor DSB1559 efficacy in BRCA1-associated triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7120.

Novel synthesized ionizable lipid for LNP-mediated P2X7siRNA to inhibit migration and induce apoptosis of breast cancer cells.

The development of ionizable lipid (IL) was necessary to enable the effective formulation of small interfering RNA (siRNA) to inhibit P2X7 receptors (P2X7R), a key player in tumor proliferation, apoptosis, and metastasis. In this way, the synthesis and utility of IL for enhancing cellular uptake of lipid nanoparticles (LNP) improve the proper delivery of siRNA-LNPs for knockdown overexpression of P2X7R. Therefore, to evaluate the impact of P2X7 knockdown on breast cancer (BC) migration and apoptosis, a branched and synthesized ionizable lipid (SIL) was performed for efficient transfection of LNP with siRNA for targeting P2X7 receptors (siP2X7) in mouse 4T-1 cells. Following synthesis and structural analysis of SIL, excellent characterization of the LNP was achieved (Z-average 126.8 nm, zeta-potential - 12.33, PDI 0.16, and encapsulation efficiency 85.35%). Afterward, the stability of the LNP was evaluated through an analysis of the leftover composition, and toxic concentration values for SIL and siP2X7 were determined. Furthermore, siP2X7-LNP cellular uptake in the formulation was assessed via confocal microscopy. Following determining the optimal dose (45 pmol), wound healing analysis was assessed using scratch assay microscopy, and apoptosis was evaluated using flow cytometry. The use of the innovative branched SIL in the formulation of siP2X7-LNP resulted in significant inhibition of migration and induction of apoptosis in 4T-1 cells due to improved cellular uptake. Subsequently, the innovative SIL represents a critical role in efficiently delivering siRNA against murine triple-negative breast cancer cells (TNBC) using LNP formulation, resulting in significant efficacy.

Inhibition of tumor migration and invasion by fenofibrate via suppressing epithelial-mesenchymal transition in breast cancers

The recurrence and metastasis in breast cancer within 3 years after the chemotherapies or surgery leads to poor prognosis with approximately 1-year overall survival. Large-scale scanning research studies have shown that taking lipid-lowering drugs may assist to reduce the risk of death from many cancers, since cholesterol in lipid rafts are essential for maintain integral membrane structure and functional signaling regulation. In this study, we examined five lipid-lowering drugs: swertiamarin, gemfibrozil, clofibrate, bezafibrate, and fenofibrate in triple-negative breast cancer, which is the most migration-prone subtype. Using human and murine triple-negative breast cancer cell lines (Hs 578 t and 4 T1), we found that fenofibrate displays the highest potential in inhibiting the colony formation, wound healing, and transwell migration. We further discovered that fenofibrate reduces the activity of pro-metastatic enzymes, matrix metalloproteinases (MMP)-9 and MMP-2. In addition, epithelial markers including E-cadherin and Zonula occludens-1 are increased, whereas mesenchymal markers including Snail, Twist and α-smooth muscle actin are attenuated. Furthermore, we found that fenofibrate downregulates ubiquitin-dependent GDF-15 degradation, which leads to enhanced GDF-15 expression that inhibits cell migration. Besides, nuclear translocation of FOXO1 is also upregulated by fenofibrate, which may responsible for GDF-15 expression. In summary, fenofibrate with anti-cancer ability hinders TNBC from migration and invasion, and may be beneficial to repurposing use of fenofibrate.

Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

Artificial Diets with Selective Restriction of Amino Acids and Very Low Levels of Lipids Induce Anticancer Activity in Mice with Metastatic Triple-Negative Breast Cancer.

Patients with metastatic triple negative breast cancer (TNBC) need new therapies to improve the low survival rates achieved with standard treatments. In this work, we show for the first time that the survival of mice with metastatic TNBC can be markedly increased by replacing their normal diet with artificial diets in which the levels of amino acids (AAs) and lipids are strongly manipulated. After observing selective anticancer activity in vitro, we prepared five artificial diets and evaluated their anticancer activity in a challenging model of metastatic TNBC. The model was established by injecting 4T1 murine TNBC cells into the tail vein of immunocompetent BALB/cAnNRj mice. First-line drugs doxorubicin and capecitabine were also tested in this model. AA manipulation led to modest improvements in mice survival when the levels of lipids were normal. Reducing lipid levels to 1% markedly improved the activity of several diets with different AA content. Some mice fed the artificial diets as monotherapy lived much longer than mice treated with doxorubicin and capecitabine. An artificial diet without 10 non-essential AAs, with reduced levels of essential AAs, and with 1% lipids improved the survival not only of mice with TNBC but also of mice with other types of metastatic cancers.

Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators.

Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.

Fn14-Directed DART Nanoparticles Selectively Target Neoplastic Cells in Preclinical Models of Triple-Negative Breast Cancer Brain Metastasis.

Triple-negative breast cancer (TNBC) patients with brain metastasis (BM) face dismal prognosis due to the limited therapeutic efficacy of the currently available treatment options. We previously demonstrated that paclitaxel-loaded PLGA-PEG nanoparticles (NPs) directed to the Fn14 receptor, termed "DARTs", are more efficacious than Abraxane─an FDA-approved paclitaxel nanoformulation─following intravenous delivery in a mouse model of TNBC BM. However, the precise basis for this difference was not investigated. Here, we further examine the utility of the DART drug delivery platform in complementary xenograft and syngeneic TNBC BM models. First, we demonstrated that, in comparison to nontargeted NPs, DART NPs exhibit preferential association with Fn14-positive human and murine TNBC cell lines cultured in vitro. We next identified tumor cells as the predominant source of Fn14 expression in the TNBC BM-immune microenvironment with minimal expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then show that despite similar accumulation in brains harboring TNBC tumors, Fn14-targeted DARTs exhibit significant and specific association with Fn14-positive TNBC cells compared to nontargeted NPs or Abraxane. Together, these results indicate that Fn14 expression primarily by tumor cells in TNBC BMs enables selective DART NP delivery to these cells, likely driving the significantly improved therapeutic efficacy observed in our prior work.

DNA minicircles as novel STAT3 decoy oligodeoxynucleotides endowed with anticancer activity in triple-negative breast cancer

Decoy technology is a versatile and specific DNA oligonucleotide-based targeting strategy of pathogenic transcription factors (TFs). Chemical modifications of linear decoy oligonucleotides have been made to decrease nuclease sensitivity because of the presence of free ends but at the cost of new limitations that affect their use as therapeutic drugs. Although a short DNA minicircle is a phosphodiester nucleic acid without free ends, its potential therapeutic activity as a TF decoy oligonucleotide has not yet been investigated. Here we describe the in vitro and in vivo activity of formulated 95-bp minicircles bearing one or several STAT3 binding sequences in triple-negative breast cancer (TNBC). Minicircles bearing one STAT3 binding site interacted specifically with the active form of STAT3 and inhibited proliferation, induced apoptosis, slowed down cell cycle progression, and decreased STAT3 target gene expression in human and murine TNBC cells. Intratumoral injection of STAT3 minicircles inhibited tumor growth and metastasis in a murine model of TNBC. Increasing the number of STAT3 binding sites resulted in improved anticancer activity, opening the way for a TF multitargeting strategy. Our data provide the first demonstration of minicircles acting as STAT3 decoys and show that they could be an effective therapeutic drug for TNBC treatment.

Combination of OX40 Co-Stimulation, Radiotherapy, and PD-1 Inhibition in a Syngeneic Murine Triple-Negative Breast Cancer Model.

Simple SummaryThis experimental study was designed in order to investigate the efficacy of the triple combination of radiation (SBRT), PD-1 blockade, and OX40 co-stimulation in a syngeneic murine model using ‘immunologically cold’ triple-negative breast cancer cells. SBRT can induce immunogenic tumor cell deaths and act as an in situ vaccine while OX40 signaling has been shown to improve anticancer immunity combined with PD-1 inhibition via multiple preclinical studies. In our study, triple combination therapy significantly improved primary/abscopal tumor control and reduced lung metastases compared to single or dual therapies. This was found to be through an increased ratio of CD8+ T cells to regulatory T cells and a reduced proportion of exhausted T cells in the tumor microenvironment.Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.

Abstract 1557: Intratumoral plasmid IL-12 enhanced the systemic anti-tumor effect of anti-PD-1 antibody in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Although immune checkpoint inhibitors (ICI) have entered the therapeutic landscape in TNBC, their benefits are limited to a minority of patients. We hypothesized the combination of ICI with the intratumoral administration of plasmid IL-12 (tavokinogene telseplasmid; TAVOTM) followed by electroporation could enhance the therapeutic efficacy based on our previous observation of the anti-tumor effect of TAVO in TNBC. The purpose of this study was to clarify the potency of TAVO as an additive local treatment to systemic anti-PD-1 immunotherapy. Methods: A murine TNBC cell line, E0771, was implanted bilaterally into flanks of female C57BL/6 mice, which were randomized into four groups: control (control plasmid + control antibody), anti-PD-1 (control plasmid + anti-PD-1), Tavo (Tavo + control antibody), and combination (TAVO + anti-PD-1). TAVO or control plasmid (50 µg/injection) was administered into a single tumor followed by electroporation on days 0, 3, and 7, as we previously reported (SITC abstract), while tumors on the other side were left untreated. Anti-PD-1 or control antibody (200 µg/injection) was administered by intraperitoneal injection on days 2, 6, 9, and 13. Tumor volume was measured every other day, and the survival of mice was monitored. The local microenvironment of both treated and untreated tumors and systemic effect on splenocytes were evaluated using flow cytometry and single-cell RNA-sequencing. Results: The combination of intratumoral administration of TAVO with anti-PD-1 suppressed the tumor growth more effectively compared to the other groups not only in treated local tumors but also in untreated remote tumors. Higher rates of tumor regression and better survival were observed in the combination group. TAVO locally increased effector CD8+ T cells while reducing regulatory T cells in both treated tumors and distant untreated tumors. The systemic expansion of effector CD8+ T cells was also observed in splenocytes. Conclusions: Intratumoral electroporation of TAVO with anti-PD-1 improved systemic anti-tumor efficacy by modifying the immunosuppressive tumor microenvironment. Clinical studies of this combination treatment in TNBC are underway. Citation Format: Hiroshi Nagata, Takuya Osada, Erika J. Crosby, David A. Canton, Chris G. Twitty, H. Kim Lyerly. Intratumoral plasmid IL-12 enhanced the systemic anti-tumor effect of anti-PD-1 antibody in triple-negative 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 1557.

10]-Gingerol-Loaded Nanoemulsion and its Biological Effects on Triple-Negative Breast Cancer Cells.

The apoptotic, cytotoxic, and cytostatic activities for [10]-gingerol in triple-negative breast cancer cells (TNBCs) were already reported. However, despite these important antitumor activities, the compound has the disadvantage to have a hydrophobic characteristic, hindering in vivo administration. To surpass this issue, in this study we have created a [10]-gingerol-loaded nanoemulsion (10GNE) in order to increase the stability and solubility of the compound. The nanoemulsion was characterized and tested for its cytotoxic, cytostatic, and apoptotic effects on a panel of murine and human TNBC cell lines, as well as non-tumor cells, and compared with a [10]-gingerol-free nanoemulsion (NE) and with [10]-gingerol itself. Except for the murine 4T1.13 cell line, the IC50 of the free 10G molecule, after 72 h of incubation, was higher in all cell lines tested, both murine and human, demonstrating therefore the efficacy of the 10GNE regarding cytotoxicity. In murine tumor cells, 60 μM 10GNE was able to arrest cell cycle at sub-G0 phase and induce apoptosis, leading to 48% and 78% of total cell death in 4T1.13 and 4T1Br4 murine tumor cells, respectively. This represents an improvement compared to 10G-free molecule that only induced 74% of total apoptosis at 100 μM in 4T1Br4 cells. Taken together, our results show that nanoformulation preserved the [10]-gingerol cytotoxic and cytostatic properties and improved its apoptotic function on murine TNBC cell lines. These data open new perspectives to a more suitable drug-delivery approach for [10]-gingerol for TNBC treatment that should be further demonstrated using in vivo assays.

Involvement of a Transcription factor, Nfe2, in Breast Cancer Metastasis to Bone.

Simple SummaryBreast cancer patients are frequently complicated by bone metastasis, which deteriorates their life expectancy. Bone metastasis is treated with the drugs targeting osteoclast activation, which is mostly observed at the metastasis sites. However, these drugs cannot directly inhibit cancer cell growth and therefore, a novel therapeutic strategy is required to impede cancer cell proliferation at bone metastasis sites. Here, we proved that a transcription factor, NFE2, was expressed selectively in breast cancer cells at bone metastasis sites and contributed crucially to their enhanced proliferation therein, by activating Wnt pathway. Thus, NFE2 can be a novel molecular target to treat breast cancer bone metastasis.Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

Abstract LB-391: Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC is urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs in African-American and Latina patients. Since hormone receptors, like insulin-like growth factor-1/insulin (IGF1R/IR, are known to be enriched and to cross-communicate in subtypes of TNBC, we assessed effects of IGF1R/IR antagonists on proliferation of human and murine TNBC cells in vitro. Combination treatment of BMS-754807 (IGF1R/IR) with NVP-AEW541 (IGF1R inhibitor) reduced proliferation of human MDA-MB-231, BT549, HCC1937 and murine 4T1 TNBC cells in vitro (P<0.001). Moreover, combination treatment using BMS-754807 and NVP-AEW541 significantly reduced MDA-MB-231 tumor xenograft progression in vivo (P<0.001). IHC study of tumor tissues from in vivo experiments confirmed reduced Ki-67 expression with combination treatments. Among breast cancer subtypes, TNBC is characterized by significant numbers of tumor infiltrating lymphocytes (TILs), a predictive marker for response to immunotherapy. Further, recent reports from clinical trials using immune checkpoint inhibitors demonstrate that a subset of TNBC patients respond to treatment with immune checkpoint inhibitors (ICIs). It is also notable that increased expression of IGF1Rs are identified on immune cells responsible for mounting adaptive and humoral immune responses. Using murine 4T1 TNBC implants in syngeneic, immune-competent BALB/c mice, we find that combination treatment with BMS-754807 and anti-PD-L1 antibody significantly blocks tumor progression as compared to both controls and with either agent used alone (P<0.0001). In addition, single cell analysis of the tumor microenvironment after treatment with BMS-754807 alone or combined with nti-PD-L1 antibody combination therapy shows a significant reduction in myeloid-derived suppressor cells (MDSCs). Moreover, BMS-754807 combined with anti-PD-L1 antibody increased infiltrating effector T cells and significantly enhanced the T central memory population (Tcm) (P<0.01). Functionally, it is reported that Tcm subsets of immune cells produce effector cytokines in response to antigens and can readily proliferate and differentiate into effector cells. Use of immunotherapy is a promising management option for a subset of TNBC patients, and combination treatments using IGF1R antagonists with immune checkpoint inhibitor may constitute a new treatment strategy to combat this deadly disease. [Funding by NCI U54 CA1433930; California Breast Cancer Research Program; UCLA Jonsson Citation Format: Nalo M. Hamilton, Diana C. Marquez-Garban, Lorena P. Burton, Begonya Comin-Anduix, Alejandro J. Garcia, Jaydutt V. Vadgama. Combination of insulin-like growth factor-1 receptor/insulin receptor (IGF1R/IR) antagonist with anti-PD-L1 antibody blocks triple-negative breast cancer (TNBC) progression [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 LB-391.