Triple-negative Breast Cancer Models Research Articles

Abstract B040: Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer

Abstract Breast cancer is one of the most frequently diagnosed cancers and the second leading cause of women’s cancer death. Although the five-year survival rate for women in the United States with nonmetastatic invasive breast cancer is 90%, this rate drops to less than 30% for those with tumors that have metastasized to distant organs. Epithelial-to-Mesenchymal Transition (EMT) plays a critical role in tumor metastasis by enabling the loss of epithelial characteristics and the gain of mesenchymal traits, leading to increased cell motility, invasion, and resistance to therapy. Consequently, targeting the EMT process may prevent tumor cell migration and improve patient outcomes. Rather than being a binary switch from an epithelial to a mesenchymal state, cells undergoing EMT can assume a spectrum of intermediate states, some of which may reshape the tumor microenvironment by recruiting pro-tumor immunosuppressive cells, such as regulatory T cells, M2-like macrophages, and myeloid-derived suppressor cells. However, the impact of EMT on the immune composition of the tumor microenvironment and the causative mechanisms remain poorly understood. Using multi-omics approaches in a mouse model of triple-negative breast cancer, we assessed the interaction between tumor and immune cells in the tumor microenvironment driven by the EMT. A heterogeneous mouse breast cancer cell line (4T1) was used to isolate five distinct single-cell-derived clonal populations residing in distinct states within the EMT spectrum. These EMT clones, along with the parental 4T1 cell line, were orthotopically transplanted into the mammary fat pads of BALB/c mice. Primary tumors were subsequently harvested and dissociated for single-cell RNA-sequencing (scRNA-seq) and DNA methylation analysis. Lungs were histologically evaluated for metastases. Tumors derived from an intermediate EMT clone exhibited increased tumor growth kinetics and a higher incidence of metastasis compared to epithelial or mesenchymal clones. In scRNA-seq data, tumors derived from different EMT clones largely retained their original EMT states in vivo and recruited distinct immune cell profiles. DNA methylation analysis further revealed that these tumors displayed unique methylation patterns in EMT-related CpGs. This study introduced a model that captures the complexity of the tumor microenvironment across the EMT spectrum. This was accomplished through immunophenotyping using scRNA-seq and identifying unique epigenetic patterns through DNA methylation. Our findings highlight the potential for distinct immune cell populations to be recruited to tumors in different EMT states, thereby influencing survival and metastasis outcomes. Furthermore, our results open new opportunities for developing cancer combination treatment strategies that target specific tumor-immune cell interactions. Citation Format: Hanxu Lu, Meisam Bagheri, Todd Miller, Diwakar Pattabiraman, Brock Christensen. Investigating the impact of tumor EMT states on immune cell infiltration in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B040.

Abstract C045: The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer cases and is notoriously aggressive, often developing resistance to standard chemotherapy thus leading to a high rate of metastatic relapse. Evidence suggests that this resistance is linked to changes in the extracellular matrix (ECM) composition, particularly an increase in periostin (POSTN) expression, which is also associated with poor prognosis in breast cancer. Our recent publication showed that the ECM alone can influence macrophage phenotypes. Furthermore, another study from our lab has demonstrated that POSTN mediates resistance to anti-angiogenic therapy by recruiting macrophages in pancreatic neuroendocrine tumors. Notably, POSTN deletion reduced monocyte/macrophage recruitment and enhanced cytotoxic CD8+ T-cell infiltration. Based on these findings, we hypothesized that paclitaxel (PTX), a widely used chemotherapeutic drug for the management of TNBC, may stimulate POSTN production by fibroblasts leading to the recruitment of pro-tumoral macrophages that facilitate tumor growth resulting into therapy resistance. In this study, we showed that PTX induces increased POSTN expression in mouse models of TNBC through immunohistochemistry, and in both 2D and 3D in vitro models via interactions between TNBC cell lines and mammary fibroblasts, as demonstrated by immunofluorescence. An in vitro migration assay confirmed that PTX-induced POSTN enhances the recruitment of human monocytes and macrophages, which display a mixed immunosuppressive and inflammatory phenotype, as revealed by flow cytometry. Not only did these recruited macrophages exhibit a reduced phagocytic capacity against labelled TNBC cells in an in vitro phagocytosis assay, but we also found that POSTN plays a crucial role in inducing SIRPα expression, that may diminish macrophage phagocytic potential. Increased sialylation and Siglec-1 expression are known to be linked to poor prognosis and polarization towards a more immunosuppressive macrophage phenotype in TNBC. Interestingly in our model, we observed that higher concentrations of recombinant human POSTN induce increased Siglec-1 expression on macrophages, a receptor that interacts with sialic acids in the tumor ECM. Further work using a decellularized tissue model that preserves only the ECM from patient-derived tumor biopsies pre- and post-chemo, aims to further explore the correlation between PTX-induced POSTN, ECM sialylation, and Siglec-1 expression on macrophages. Additionally, we have developed POSTN-knockout murine cancer cells and fibroblasts using CRISPR/Cas9, allowing us to investigate how POSTN influences macrophage phenotypes and chemotherapy responses in vivo. Understanding the mechanisms behind chemotherapy resistance in TNBC is crucial for developing effective treatments for this aggressive cancer subtype. By elucidating the intricate interplay between POSTN, macrophages, and chemotherapy response, we aim to identify new therapeutic strategies to improve outcomes for TNBC patients. Citation Format: Ludovica Tarantola, Ioanna Keklikoglou, Oliver M. Pearce. The extracellular matrix glycoprotein periostin supports chemotherapy resistance by modulating macrophage recruitment and phagocytosis in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C045.

Insulin-like growth factor-binding protein-3 is induced by tamoxifen and fulvestrant and modulates fulvestrant response in breast cancer cells

IntroductionInsulin-like growth factor binding protein-3 (IGFBP-3) exerts varying effects on estrogen receptor alpha (ERα)-positive and triple-negative breast cancer (TNBC) cells. In ERα-positive cells, IGFBP-3 is antiproliferative and proapoptotic. In contrast, IGFBP-3 stimulates proliferation in triple-negative breast cancer (TNBC) cells via EGFR activation.MethodsTo identify potential mechanisms that underlie the opposing effects of IGFBP-3 on these two breast cancer subtypes, IGFBP-3 expression was determined in cell line models of both ERα-positive breast cancer and TNBC, and cells were treated with antiestrogens tamoxifen and fulvestrant. Results and discussionMCF-7 and T-47D cells expressed low levels of IGFBP-3 when compared to MDA-MB-231 and MDA-MB-468 cells. MCF-7 cells with acquired resistance to the selective estrogen receptor degrader fulvestrant expressed high IGFBP-3 and MCF-7 cells with constitutive IGFBP-3 expression were fulvestrant resistant. IGFBP-3 expression was increased in all cell lines upon treatment with fulvestrant or the selective estrogen receptor modulator tamoxifen and both fulvestrant and tamoxifen increased TNBC cell proliferation. Further, IGFBP-3 expression was increased by treatment with the GPER1 agonist G-1 and attenuated upon treatment with P17, a YAP/TAZ inhibitor. These data suggest that IGFBP-3 modulates breast cancer cells and is a mediator of breast cancer cell response to fulvestrant and tamoxifen.

Mediator kinase inhibitors suppress triple-negative breast cancer growth and extend tumor suppression by mTOR and AKT inhibitors

Triple-negative breast cancers (TNBC) are treated primarily by chemotherapy and lack clinically validated therapeutic targets. In particular, inhibitors of the PI3K/AKT/mTOR pathway, abnormally activated in many breast cancers, failed to achieve clinical efficacy in TNBC due to the development of adaptive drug resistance, which is largely driven by the transcriptomic plasticity of TNBC. Expression of CDK8/19 Mediator kinases that control transcriptional reprogramming correlates with relapse-free survival and treatment failure in breast cancer patients, including TNBC. We now investigated how CDK8/19 inhibitors affect the growth of TNBC tumors and their response to mTOR and AKT inhibitors. In contrast to the effects of most anticancer drugs, all the tested human TNBC models (including patient-derived xenografts) responded to CDK8/19 inhibitors in vivo even when they did not respond in vitro. Furthermore, CDK8/19 inhibition extended the host survival of established lung metastases in a murine TNBC model, where the primary tumors were not significantly affected. CDK8/19 inhibitors synergized with an mTORC1 inhibitor everolimus and a pan-AKT inhibitor capivasertib in vitro and strongly potentiated these drugs in long-term in vivo studies. Transcriptomic analysis of tumors that responded or became adapted to everolimus revealed that drug adaptation in vivo was associated with major transcriptional changes in both tumor and stromal cells. Combining everolimus with a CDK8/19 inhibitor counteracted many of these changes and induced combination-specific effects on the expression of multiple genes that affect tumor growth. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors as a new type of drugs for TNBC therapy.

DNA tetrahedral nanoparticles: Co-delivery of siOTUD6B/DOX against triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options. Recently, the deubiquitinizing enzyme ovarian tumor domain-containing 6B (OTUD6B) has been reported to play a potential role in TNBC progression. Therefore, this study investigates the role and underlying molecular mechanisms of OTUD6B in vitro and xenograft models of TNBC. Specifically, we examined the therapeutic effects of siOTUD6B and doxorubicin (DOX) co-delivery using synthesized tetrahedral DNA nanoparticles (Tds) on tumor growth and progression. Additionally, the uptake and efficacy of the siOTUD6B/DOX@Td in TNBC cells were evaluated. Notably, the siOTUD6B/DOX@Td nanoparticle demonstrated efficient cellular uptake by TNBC cells, resulting in OTUD6B knockdown and controlled release of DOX. Additionally, siOTUD6B/DOX@Td treatment enhanced apoptosis rates increased DOX sensitivity, and inhibited TNBC cell growth, migration, and metastasis. Moreover, in vivo experiments confirmed that siOTUD6B/DOX@Td treatment inhibited tumor growth and metastasis without damaging the primary organs. Mechanistically, OTUD6B regulates TNBC progression by stabilizing murine double minute 2 (MDM2) and degrading forkhead box O3a (FOXO3a). Conclusively, this study demonstrates the potential applicability of DNA nanoparticles loaded with DOX and siOTUD6B for TNBC treatment.

Cationic Metal-Organic Layer Delivers siRNAs to Overcome Radioresistance and Potentiate Cancer Radiotherapy.

Radiotherapy plays an important role in modern oncology, but its treatment efficacy is limited by the radioresistance of tumor cells. As a member of the inhibitor of apoptosis protein family, survivin plays a key role in developing radioresistance by mediating apoptosis evasion, promoting epithelial-mesenchymal transition, and modulating cell cycle dynamics. Efficient downregulation of survivin expression presents a promising strategy to enhance the antitumor effects of radiotherapy. Herein, we report the design of a hafnium-porphyrin-based cationic metal-organic layer (CMOL) with quaternary ammonium capping groups to deliver small interfering RNAs (siRNAs) for enhanced radiotherapy. The CMOL@siRNA nanoplatform not only increased energy deposition from X-rays and reactive oxygen species generation via a unique radiotherapy-radiodynamic therapy process, but also effectively delivered siRNAs to downregulate survivin expression and ameliorate radioresistance of cancer cells. Consequently, CMOL@siRNA in combination with low-dose X-ray irradiation demonstrated remarkable antitumor efficacy with 96.9% and 91.4% tumor growth inhibition in murine colorectal carcinoma and triple-negative breast cancer models, respectively.

MicroRNAome profiling of breast cancer unveils hsa-miR-5683 as a tumor suppressor microRNA predicting favorable clinical outcome.

Breast cancer is a heterogeneous disease with diverse molecular subtypes, underscoring a better understanding of its molecular features and underlying regulatory mechanisms. Therefore, identifying novel prognostic biomarkers and therapeutic targets is crucial for advancing the current standard of care for breast cancer patients. Ninety-six formalin-fixed paraffin-embedded (FFPE) breast cancer samples underwent miRNAome profiling using QIAseq microRNA library kit and sequencing on Illumina platform. Mature miRNA quantification was conducted using CLC Genomics Workbench v21.0.5, while Relapse-free survival (RFS) analysis was conducted using RStudio 2023.09.1. Gain-of-function studies were conducted using miRNA mimics, while the effects of miRNA exogenous expression on cancer hallmark were assessed using 2-dimentional (2D) proliferation assay, three-dimensional (3D) organotypic culture, and live-dead staining. TargetScan database and Ingenuity Pathway Analysis (IPA) were used for miRNA target identification. Hierarchical clustering based on miRNA expression revealed distinct patterns in relation to PAM50 classification and identified miRNAs panels associated with luminal, HER2, and basal subtypes. hsa-miR-5683 emerged as a potential prognostic biomarker, showing a favorable correlation with RFS and suppressing tumorigenicity under 2D and 3D conditions in triple-negative breast cancer (TNBC) models. Findings were further extended to the MCF7 hormone receptor positive (HR+) model. Transcriptomic profiling of hsa-miR-5683 overexpressing TNBC cells revealed its potential role in key oncogenic pathways. Integration of downregulated genes and CRISPR-Cas9 perturbational effects identified ACLY, RACGAP1, AK4, MRPL51, CYB5B, MKRN1, TMEM230, NUP54, ANAPC13, PGAM1, and SOD1 as bona fide gene targets for hsa-miR-5683. Our data provides comprehensive miRNA expression atlas in breast cancer subtypes and underscores the prognostic and therapeutic significance of numerous miRNAs, including hsa-miR-5683 in TNBC. The identified gene targets unravel the intricate regulatory network in TNBC progression, suggesting promising avenues for further research and targeted therapeutic interventions.

Inhibition of GPX4 enhances CDK4/6 inhibitor and endocrine therapy activity in breast cancer.

CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. Here, we perform two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulate sensitivity, with GPX4 as the top sensitiser in both screens. Depletion or inhibition of GPX4 increases sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitises triple negative breast cancer (TNBC) models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation is promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer.

Single-cell sequencing unveils mitophagy-related prognostic model for triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking hormone receptors and HER2 expression, leading to limited treatment options and poor prognosis. Mitophagy, a selective autophagy process targeting damaged mitochondria, plays a complex role in cancer progression, yet its prognostic significance in TNBC is not well understood.MethodsThis study utilized single-cell RNA sequencing data from the TCGA and GEO databases to identify mitophagy-related genes (MRGs) associated with TNBC. A prognostic model was developed using univariate Cox analysis and LASSO regression. The model was validated across multiple independent cohorts, and correlations between MRG expression, immune infiltration, and drug sensitivity were explored.ResultsNine key MRGs were identified and used to stratify TNBC patients into high-risk and low-risk groups, with the high-risk group showing significantly worse survival outcomes. The model demonstrated strong predictive accuracy across various datasets. Additionally, the study revealed a correlation between higher MRG expression levels and increased immune cell infiltration, as well as potential responsiveness to specific chemotherapeutic agents.ConclusionThe mitophagy-related prognostic model offers a novel method for predicting outcomes in TNBC patients and highlights the role of mitophagy in influencing the tumor microenvironment, with potential applications in personalized treatment strategies.

ICOS-expressing CAR-T cells mediate durable eradication of triple-negative breast cancer and metastasis

BackgroundThe failure of conventional therapies and the propensity for recurrence and metastasis make triple-negative breast cancer (TNBC) a formidable challenge with grim prognoses and diminished survival rates. Immunotherapy, including immune...

Evaluation of Targeted Alpha Therapy Using [211At]FAPI1 in Triple-Negative Breast Cancer Xenograft Models.

Triple-negative breast cancer (TNBC) presents limited therapeutic options and is associated with poor prognosis. Early detection and the development of novel therapeutic agents are therefore imperative. Fibroblast activation protein (FAP) is a membrane protein expressed on cancer-associated fibroblasts (CAFs) that plays an essential role in TNBC proliferation, migration, and invasion. Consequently, it is hypothesized that the Astatine (211At)-labeled FAP inhibitor (FAPI) selectively exerts anti-tumor effects through alpha-particle emission. In this study, we aimed to assess its theranostic capabilities by integrating [18F]FAPI-74 PET imaging with targeted alpha therapy using [211At]FAPI1 in TNBC models. Mice xenografts were established by transplanting MDA-MB-231 and HT1080 cells (control). As a parallel diagnostic method, [18F]FAPI-74 was administered for PET imaging to validate FAP expression. A single dose of [211At]FAPI1 (1.04 ± 0.10 MBq) was administered to evaluate the therapeutic efficacy. [18F]FAPI-74 exhibited high accumulation in MDA-MB-231 xenografts, and FAP expression was pathologically confirmed via immunostaining. The group that received [211At]FAPI1 (n = 11) demonstrated a significantly enhanced anti-tumor effect compared with the control group (n = 7) (p = 0.002). In conclusion, [18F]FAPI-74 PET imaging was successfully used to diagnose FAP expression, and as [211At]FAPI1 showed promising therapeutic efficacy in TNBC models, it is expected to be a viable therapeutic option.

Virtual patient analysis identifies strategies to improve the performance of predictive biomarkers for PD-1 blockade

Patients with metastatic triple-negative breast cancer (TNBC) show variable responses to PD-1 inhibition. Efficient patient selection by predictive biomarkers would be desirable but is hindered by the limited performance of existing biomarkers. Here, we leveraged in silico patient cohorts generated using a quantitative systems pharmacology model of metastatic TNBC, informed by transcriptomic and clinical data, to explore potential ways to improve patient selection. We evaluated and quantified the performance of 90 biomarker candidates, including various cellular and molecular species, at different cutoffs by a cutoff-based biomarker testing algorithm combined with machine learning-based feature selection. Combinations of pretreatment biomarkers improved the specificity compared to single biomarkers at the cost of reduced sensitivity. On the other hand, early on-treatment biomarkers, such as the relative change in tumor diameter from baseline measured at two weeks after treatment initiation, achieved remarkably higher sensitivity and specificity. Further, blood-based biomarkers had a comparable ability to tumor- or lymph node-based biomarkers in identifying a subset of responders, potentially suggesting a less invasive way for patient selection.

A Machine Learning-Optimized System for Pulsatile, Photo- and Chemotherapeutic Treatment Using Near-Infrared Responsive MoS2-Based Microparticles in a Breast Cancer Model.

Multimodal cancer therapies are often required for progressive cancers due to the high persistence and mortality of the disease and the negative systemic side effects of traditional therapeutic methods. Thus, the development of less invasive modalities for recurring treatment cycles is of clinical significance. Herein, a light-activatable microparticle system was developed for localized, pulsatile delivery of anticancer drugs with simultaneous thermal ablation by applying controlled ON-OFF thermal cycles using near-infrared laser irradiation. The system is composed of poly(caprolactone) microparticles of 200 μm size containing molybdenum disulfide (MoS2) nanosheets as the photothermal agent and hydrophilic doxorubicin or hydrophobic violacein, as model drugs. Upon irradiation, the nanosheets heat up to ≥50 °C leading to polymer softening and release of the drug. MoS2 nanosheets exhibit high photothermal conversion efficiency and require low-power laser irradiation. A machine learning algorithm was applied to acquire the optimal laser operation conditions. In a mouse subcutaneous model of 4T1 triple-negative breast cancer, 25 microparticles were intratumorally administered, and after 3-cycle laser treatment, the system conferred synergistic phototherapeutic and chemotherapeutic effects. Our on-demand, pulsatile synergistic treatment resulted in increased median survival up to 39 days post start of treatment compared to untreated mice, with complete eradication of the tumors at the primary site. Such a system is therapeutically relevant for patients in need of recurring cycles of treatment on small tumors, since it provides precise localization and low invasiveness and is not cross-resistant with other treatments.

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.

AKT and EZH2 inhibitors kill TNBCs by hijacking mechanisms of involution.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and has the highest rate of recurrence1. The predominant standard of care for advanced TNBC is systemic chemotherapy with or without immunotherapy; however, responses are typically short lived1,2. Thus, there is an urgent need to develop more effective treatments. Components of the PI3K pathway represent plausible therapeutic targets; more than 70% of TNBCs have alterations in PIK3CA, AKT1 or PTEN3-6. However, in contrast to hormone-receptor-positive tumours, it is still unclear whether or how triple-negative disease will respond to PI3K pathway inhibitors7. Here we describe a promising AKT-inhibitor-based therapeutic combination for TNBC. Specifically, we show that AKT inhibitors synergize with agents that suppress the histone methyltransferase EZH2 and promote robust tumour regression in multiple TNBC models in vivo. AKT and EZH2 inhibitors exert these effects by first cooperatively driving basal-like TNBC cells into a more differentiated, luminal-like state, which cannot be effectively induced by either agent alone. OnceTNBCs are differentiated, these agents kill them by hijacking signals that normally drive mammary gland involution. Using a machine learning approach, we developed a classifier that can be used to predict sensitivity. Together, these findings identify a promising therapeutic strategy for this highly aggressive tumour type and illustrate how deregulated epigenetic enzymes can insulate tumours from oncogenic vulnerabilities. These studies also reveal how developmental tissue-specific cell death pathways may be co-opted for therapeutic benefit.

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

Photocatalytic scaffolds enhance anticancer performances of bacterial consortium AUN

Microbial metabolism and function are regulated by ambient microenvironment, yet no functional microporous materials are known to regulate anticancer activity of complex bacterial systems. Here we show that a simple scaffold-mediated bacterial culture is useful for enhancing the anticancer therapeutic abilities of a microbial consortium designated as AUN and composed of intratumoural bacteria Proteus mirabilis (A-gyo) and purple photosynthetic bacteria Rhodopseudomonas palustris (UN-gyo). Our discovery of the mechanism by which porous scaffolds influence the bacterial activities of AUN will facilitate further designs of artificial scaffold material for effective treatment against drug-resistant, triple-negative breast cancer models. A polydimethylsiloxane composite artificial scaffold encapsulating light-activatable TiO2 particularly enhanced the therapeutic outcomes of AUN in syngeneic and orthotopic models. These strong anticancer efficacies were synergistically expressed in tumours because of the potent oncolytic ability of AUN with the help of various immune cells. AUN administration to canines showed well tolerability in an exploratory study.

Enhanced antitumor activity of lapatinib against triple-negative breast cancer via loading in human serum albumin

Triple-negative breast cancer (TNBC) presents significant treatment challenges due to its aggressive nature. Human serum albumin (HSA) is a promising drug delivery platform, offering high binding capacity, biocompatibility, and reduced toxicity. Lapatinib (LAP), a tyrosine kinase inhibitor for TNBC, is hindered by poor water solubility and toxicity. To address these issues, LAP was encapsulated within HSA (HSA-LAP), and its structural, drug release, and therapeutic properties were evaluated in cellular and animal TNBC models. HSA-LAP demonstrated efficient drug loading and pH-dependent tumor-targeted release, favoring acidic tumor microenvironments. Structural and microscopic studies confirmed LAP binding to HSA, with only minor structural and no significant morphological changes observed. In 4 T1 breast cancer cells, HSA-LAP exhibited superior anti-proliferative, pro-apoptotic, and anti-migratory effects compared to free LAP, which were further amplified when combined with VGB3, a VEGFR1/2-targeting peptide, indicating an effective dual-targeting strategy for TNBC. In vivo, HSA-LAP showed greater tumor inhibition and improved survival rates, especially in combination with VGB3 through apoptosis induction. Biodistribution studies using technetium-99 m (99mTc) labeling revealed enhanced tumor targeting. These findings highlight the potential of HSA as a delivery vehicle for LAP, particularly in combination with anti-angiogenic agents like VGB3, offering a promising therapeutic strategy for TNBC.