Potential Of Triple-negative Breast Cancer Research Articles

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

Abstract PO3-24-03: Cisplatin synergizes immune checkpoint blocking through RNFT1 mediated PD-L1 recycling in Triple Negative Breast Cancer

Abstract Since chemotherapy showed synergies with PD-1/PD-L1 blockage therapy in solid tumors, the efficacy in triple negative breast cancer (TNBC) varies greatly and only a subset of patients achieves durable responses. To increase their clinical efficacy, it is important to explore the underlying mechanisms of immune checkpoint ligand PD-L1. Here, we found cisplatin influenced the controlled intracellular transport of PDL1 by inducing the RNFT1-mediated endoplasmic reticulum autophagy. Overexpression of RNFT1 increases the endocytosis-recycling rate of the plasm membrane PD-L1, which is regulated by K63-polyubiquitination and endoplasmic reticulum autophagy degradation of PD-L1, thereby having negative impact on antitumor immunotherapy, such as reduced effective drug concentration and avoiding immune surveillance. Genetically or pharmacologically modulating RNFT1-induced K63 polyubiquitination blocks endocytosis of PD-L1, consequently enhances the antitumor response to PD-L1 blockade. Thus, our results suggest that cisplatin treatment is involved in RNFT1-induced K63 polyubiquitination of PD-L1 endocytosis-recycling pathway that governs immune response, and blocking RNFT1 might enhance the efficacy of PD-1/PD-L1 blockade. These studies have high application and translational potential in triple-negative breast cancer. Citation Format: Jiahui Chu, Yifan Zhang, Yijia Hua, Jue Gong, Jie Mei, Ziyi Fu, Yongmei Yi. Cisplatin synergizes immune checkpoint blocking through RNFT1 mediated PD-L1 recycling in Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-03.

Abstract 1518: Understanding the role of VISTA in regulating T cells function and the therapeutic potential of blocking VISTA in triple negative breast cancer

Abstract Immune checkpoint inhibitors (ICIs) targeting immune modulatory molecules have been widely used and have shown therapeutic efficacy in treating multiple malignancies in the last decade. However, in triple-negative breast cancer (TNBC), only around 5 % of patients showed a durable response to ICIs. While the response rates were increased up to 10% by combining ICIs with conventional chemotherapy, the majority of the patients did not respond to the treatment. Thus, there is a need to improve our understanding of the mechanism underlying unequal response to ICIs in TNBC and develop/discover new treatments to improve patient outcomes. The lack of response to ICIs has been attributed to the increased infiltration of immunosuppressive cells (including MDSCs, neutrophils and TAMs), as well as the presence of additional immune checkpoint receptors/ligands that may further suppress cytotoxic T cells (CD8+ T cells) in the tumor microenvironment (TME). V-domain Ig suppressor of T-cell activation (VISTA) is one of the immune checkpoint molecules attracting increasing attention in the context of anti-tumor immunity due to its broad expression across various cancer types and increased expression upon development of immune checkpoint resistance. However, the role of VISTA in the anti-tumor immune response and its therapeutic potential in TNBC remains unclear. In our study, we observed that VISTA is expressed in TNBC patients, with higher expression in neutrophils and macrophages. In the Py230 murine breast cancer model, VISTA expression increased upon paclitaxel (PTX), anti-PD-L1 and paclitaxel combined with anti-PD-L1 treatment compared to the control group, suggesting its potential role as a resistance mechanism in further inhibiting T cells. In the Py230 model, VISTA blockade increased the infiltration and activation of CD8+ T cells, as evidenced by the higher number of CD8+ T cells, increased CD69+CD8+ T cells, and Granzyme B production, indicating that VISTA blockade improved CD8+ T cells mediated anti-tumor immunity. To functionally test the role of VISTA, we stimulated CD8+ T cells and co-cultured them with naïve macrophages or tumor educated macrophages in the presence or absence of VISTA blocking antibody. Tumor-educated macrophages showed increased immunosuppressive activity which is abrogated with VISTA blockade. Moreover, we found that the immunosuppressive effect of macrophages on T cell activation needs direct cell interaction between VISTA on macrophages and T cells. In conclusion, this study shows the mechanism of action of VISTA in the regulation of T cells mediated anti-tumor immune response, and provides the rationale for blocking VISTA in TNBC. Citation Format: Maidinaimu Abudula, Yuliana Astuti, Meirion Raymant, Teifion Luckett, Patrick Freeman, Michael Schmid, Ainhoa Mielgo Iza. Understanding the role of VISTA in regulating T cells function and the therapeutic potential of blocking VISTA in 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 1518.

Abstract 5945: Exploring the antiproliferative potential of KHS101 on triple negative breast cancer cell lines

Abstract Breast cancer is the second leading cause of cancer deaths in the world. Among its subtypes, triple-negative breast cancer (TNBC) is highly aggressive, particularly affecting African American (AA) women, with limited treatment choices. HSP60 protein promotes tumorigenesis and is being studied as a possible biomarker to treat cancers such as TNBC. The small molecule KHS101 specifically binds and inhibits the mitochondrial chaperone HSP60. In this study, we hypothesized that KHS101 would inhibit the proliferation of TNBC cells and therefore may have a therapeutic potential.We studied the antiproliferative effects of KHS101 on TNBC cell lines (MDA-MB-231, MDA-MB-468, HCC1806) through MTT cell proliferation and colony forming assays at varying doses and time points (24, 48, 72, and 96 hours). We performed cell cycle analysis to understand the cell cycle response and apoptotic events in treated or control cells. Additionally, the gene expression and frequently altered pathways resulting from KHS101 treatment were analyzed by RNA sequencing of cells treated with vehicle (DMSO) or with various concentrations of KHS101(1, 2.5, 5, and 7.5 uM) for 72 hours. We analyzed the data using GraphPad Prism and Microsoft Excel, with statistical significance set at p ≤ 0.05. TNBC cell lines exhibited significant dose-response and antiproliferative effects with increased KHS101 doses and treatment durations. Notably, AA breast cancer cells (MDA-MB-468) displayed heightened cytotoxicity and reduced proliferation compared to Caucasian American (CA) breast cancer cells (MDA-MB-231 and HCC1806). Cell cycle analysis revealed G2/M phase arrest, consistent with RNA sequencing data showing downregulation of the activation of ATM/ATR response in KHS101-treated cell lines compared to the controls. Gene set enrichment analysis indicated downregulation of the cell cycle control genes in MDA-MB-231 cells after 72 hours of KHS101 treatment (7.5 µM). Reactome pathway analysis in KHS101-treated MDA-MB-231 cells showed upregulation of IFN-gamma signaling and degradation of extracellular matrix. Similarly, Kegg pathway analysis showed upregulation in cell adhesion molecules, cytokine-cytokine receptor interaction, antigen processing and presentation, and Natural Killer cell-mediated cytotoxicity in KHS101-treated cancer cells compared to the controls. KHS101 demonstrates varying cytotoxic effects on TNBC cells, with potential implications for antitumor immune responses. Our findings highlight the distinct susceptibility of AA breast cancer cells and suggest KHS101 as a promising avenue for further research in understanding its mechanism of action and validating therapeutic potential in TNBC. Citation Format: Reshma Gurung, Alehegne W. Yirsaw, Muhammad G. Khodary, Temesgen Samuel, Maninder Sandey, Clayton C. Yates, Deepa Bedi. Exploring the antiproliferative potential of KHS101 on triple negative breast cancer cell lines [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 5945.

Precise nano-system-based drug delivery and synergistic therapy against androgen receptor-positive triple-negative breast cancer

Targeting androgen receptor (AR) has shown great therapeutic potential in triple-negative breast cancer (TNBC), yet its efficacy remains unsatisfactory. Here, we aimed to identify promising targeted agents that synergize with enzalutamide, a second-generation AR inhibitor, in TNBC. By using a strategy for screening drug combinations based on the Sensitivity Index (SI), we found that MK-8776, a selective checkpoint kinase1 (CHK1) inhibitor, showed favorable synergism with enzalutamide in AR-positive TNBC. The combination of enzalutamide and MK-8776 was found to exert more significant anti-tumor effects in TNBC than the single application of enzalutamide or MK-8776, respectively. Furthermore, a nanoparticle-based on hyaluronic acid (HA)-modified hollow-manganese dioxide (HMnO2), named HMnE&M@H, was established to encapsulate and deliver enzalutamide and MK-8776. This HA-modified nanosystem managed targeted activation via pH/glutathione responsiveness. HMnE&M@H repressed tumor growth more obviously than the simple addition of enzalutamide and MK-8776 without a carrier. Collectively, our study elucidated the synergy of enzalutamide and MK-8776 in TNBC and developed a novel tumor-targeted nano drug delivery system HMnE&M@H, providing a potential therapeutic approach for the treatment of TNBC.

Curcumin suppresses metastasis of triple-negative breast cancer cells by modulating EMT signaling pathways: An integrated study of bioinformatics analysis.

This study aimed to use bioinformatics approaches for predicting the anticancer mechanisms of curcumin on triple-negative breast cancer (TNBC) and to verify these predictions through in vitro experiments. Initially, the Cell Counting Kit-8 (CCK8) assay was employed to rigorously investigate the influence of curcumin on the proliferative capacity of TNBC cells. Subsequently, flow cytometry was employed to meticulously assess the impact of curcumin on cellular apoptosis and the cell cycle regulation. Transwell assays were employed to meticulously evaluate the effect of curcumin on the motility of TNBC cells. RNA sequencing was conducted, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of differentially expressed genes, aiming to elucidate the potential anticancer mechanisms underlying curcumin's effects. To thoroughly elucidate the interactions among multiple proteins, we constructed a protein-protein interaction (PPI) network. Finally, the expression levels of several key proteins, including fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6, were assessed using the western blot. The CCK8 assay results showed that curcumin significantly inhibited the proliferation of Hs578T and MDA-MB-231 cells. Flow cytometry results showed that curcumin induced apoptosis in these cells and arrested the cell cycle at the G2/M phase. Additionally, Transwell assay results showed that curcumin effectively reduced the motility of Hs578T and MDA-MB-231 cells. Enrichment analysis of RNA sequencing data showed that the mechanism of action of curcumin was significantly associated with signaling pathways such as pathways in cancer, focal adhesion, and PI3K-Akt signaling pathways. Subsequently, we constructed a protein-protein interaction network to elucidate the interactions among multiple proteins. Finally, Western blotting analysis showed that curcumin significantly decreased the expression levels of key proteins including Fibronectin, mTOR, β-Catenin, p-Akt, Akt, N-Cadherin, p-S6, and S6. Curcumin exhibits its therapeutic potential in TNBC by modulating multiple signaling pathways. It may inhibit the epithelial-mesenchymal transition process by downregulating the expression of proteins involved in the mTOR and PI3K-Akt signaling pathways, thereby suppressing the motility of TNBC cells. These findings provide experimental evidence for considering curcumin as a potential therapeutic strategy in the treatment of TNBC.

Fucoidan-mediated targeted delivery of dasatinib-loaded nanoparticles amplifies apoptosis and endows cytotoxic potential in triple-negative breast cancer

Dasatinib (DST) is a tyrosine kinase inhibitor with established antiproliferative activity in Triple-negative breast cancer. Conventional treatment strategies with DST have several pitfalls related to the development of resistance, lower cellular uptake and unwanted adverse effects. To address these issues, we have prepared P-selectin-targeted nanoparticles of DST with fucoidan (FUC) as a ligand. Poly lactide-co-glycolide nanoparticles of DST were coated with chitosan (CH) and FUC via electrostatic interaction (DST-CH-FUC-NPs). The mean particle size of 210.36 ± 0.66 nm and a polydispersity index of 0.234 ± 0.013 was observed for DST-CH-FUC-NPs. TEM and FTIR analysis proved CH coating followed by an FUC layer on nanoparticles. DST-CH-FUC-NPs showed a sustained release profile up to 120 h and 2.9 times less hemolytic potential than free DST suspension. DST-CH-FUC-NPs demonstrated 8-fold higher cytotoxicity compared to free DST in MDA-MB-231 cells. Rhodamine-CH-FUC- NPs showed 19 times and 3 times higher cellular uptake than free Rhodamine and Rhodamine-CH-NPs, respectively. DST-CH-FUC-NPs also displayed increased ROS production and mitochondrial membrane potential damage. Apoptosis study revealed a 7.5-fold higher apoptosis index for DST-CH-FUC-NPs than free DST. Subsequently, the DST-CH-FUC-NPs showed increased inhibition of cell migration, where approximately 5 % wound closure was noted. Further, DST-CH-FUC-NPs confirmed higher disruption of lysosomal membrane integrity, which is well correlated with apoptosis results. In addition, developed NPs were nontoxic on MCF 10 A normal cells. All these findings suggest that fabricated DST-CH-FUC-NPs are promising biocompatible carriers for tumor-targeted delivery and enhanced efficacy of dasatinib.

Abstract 4037: Doxazosin exerts anti-metastatic potential in triple-negative breast cancer via impairment of cancer stem-like features

Abstract Background and Purpose: Triple-negative breast cancer (TNBC) tumors typically harbor a high cancer stem-like population leading to chemo-resistance, recurrence, and metastasis. Tumor metastasis is associated with 90% of cancer-related deaths, highlighting the urgent clinical unmet need. Doxazosin is known to inhibit cell migration and invasion in several cancer cell types; however, the precise mechanisms underlying doxazosin’s anticancer effects in TNBC have not been fully elucidated. In the present study, we sought to investigate the mechanism of action of doxazosin responsible for its effects on apoptosis, cancer stem cell (CSC)-like properties, cell migration, and metastasis in TNBC. Experimental designs: Doxazosin on TNBC cell lines [MDA-MB-231, BT549, and 4T1] in vitro was evaluated in cell viability, apoptosis, cell migration, CD44/CD24 staining, ALDH1 activity, and mammosphere formation. The effect of doxazosin on tumor growth, angiogenesis, and metastasis was evaluated in an orthotopic allograft mice model with CSC-enriched population. Results: Doxazosin significantly reduced cell viability and induced apoptosis in MDA-MB-231 and BT549 cells via activation of caspase-3/-7 and cleavage of PARP. Doxazosin significantly suppressed cell migratory capability, concomitant with disrupting cytoskeletal proteins, including vimentin and F-actin expression in TNBC cells. An impairment of BCSC-like properties was associated with reduction of ALDH1 activity and the CD44+/CD24- population, concomitant with suppression of mammosphere-forming ability. Doxazosin administration reduced tumor growth and lung metastasis, as evidenced by a sharp decline in bioluminescence signal intensity. Inhibitory effect of tumor growth was accompanied by a significant decrease of Ki-67 and enhancement of apoptosis with DNA fragmentation and increased cleaved-caspase-3 expression. The latter phenomenon was associated with the impediment of JAK2/STAT3 signaling pathway and CSC-like properties. Furthermore, no toxic effects of doxazosin were found in liver and kidney function in animals. Conclusion: Taken together, our findings highlight doxazosin as a promising candidate for drug repurposing in suppressing metastatic TNBC. Citation Format: Seongjae Kim, Dongmi Ko, Juyeon Seo, Soeun Park, Minsu Park, Kee Dal Nam, Yong koo Kang, So Ra Seock, Jaeyoun Park, Eunhye Oh, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. Doxazosin exerts anti-metastatic potential in triple-negative breast cancer via impairment of cancer stem-like features. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4037.

Exogenous hydrogen sulfide (H2S) exerts therapeutic potential in triple-negative breast cancer by affecting cell cycle and DNA replication pathway

Triple negative breast cancer (TNBC) is a highly aggressive subtype with a poor prognosis due to its high rates of proliferation and metastasis. Recently, hydrogen sulfide (H2S) has been recognized as a novel gasotransmitter that plays a significant role in various pathological processes, including cancer. Here, we show that exogenous H2S inhibited TNBC cancer cell proliferation, migration and invasion in vitro, and also decreased cancer malignances in the mouse model of TNBC. To investigate the underlying mechanisms of H2S's anti-cancer effects in TNBC, we performed transcriptome sequencing and bioinformatic analyses. 2121 differentially expressed genes (DEGs) were revealed, and mainly enriched in cell cycle and DNA replication pathways. Further analysis revealed changes in alternative splicing after exogenous H2S treatment. Protein-protein interaction (PPI) network analysis was performed, which identified 458 interactions among 276 DEGs enriched in cell cycle and DNA replication pathways.We identified seven hub genes (MCM3, MCM4, MCM5, MCM6, CDC6, CDC45, and GINS2) through PPI network analysis, which were up-regulated in clinical human breast cancer but down-regulated after H2S treatment. Based on the hub genes selected, we developed a model predicting that exogenous H2S mainly exerts its anti-TNBC role by delaying DNA replication. Our findings suggest that exogenous H2S has potential as a therapeutic agent in TNBC and may exert its therapeutic potential through DNA replication and the cell cycle pathway.

Abstract P6-10-19: Screening of GPCR-targeting drugs for repositioning in breast cancer

Abstract Drug repositioning can overcome both substantial costs and the long process of new drug discovery and development in cancer treatment. Some FDA-approved drugs have been found to have the potential to be repositioned as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repositioning potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repositioning potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of anti-HER2 therapies. We identified 7 drugs/compounds as potential hits, out of which 4 were FDA-approved drugs. We focused on beta-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide (NO) production. Nebivolol reduced invasion and migration potentials which may suggest its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology, and End Results (SEER)-Medicare dataset found a reduced but not statistically significant risk of all-cause mortality in the nebivolol group. In-depth future analyses including detailed in vivo studies and real-world data analysis with more patients are needed to investigate further the potential of nebivolol as a repositioned therapy for BC. Citation Format: Noor Abdulkareem, Raksha Bhat, Reid Powell, Soumya Chikermane, Soham Yande, Lisa Trinh, Hala Abdelnasser, Alexis Ruiz, Mary Sobieski, Nghi Nguyen, Camille Johnson, Michael Johnson, Clifford Stephan, Meghana Trivedi. Screening of GPCR-targeting drugs for repositioning in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-19.

Abstract P4-02-19: Development of a Novel Imaging Biomarker to Ascertain Responsiveness to Immunotherapy

Abstract Background: Immunotherapy has emerged as an essential treatment modality for enhancing survival in triple negative breast cancer (TNBC). Despite demonstrated improvements in pathologic complete response (pCR), both toxicity and adverse events from immuno-oncology (IO) drugs remain a significant limitation. Currently, a lack of tests to differentiate patients likely to respond to IO vs. poor responders precludes a tailored approach to immunotherapy. Here we describe an imaging biomarker that allows physicians to target breast cancer patients with the highest likelihood of response to immunotherapies. Methods: We identified a rapidly assessable, non-invasive biomarker of tumor response to immunotherapy. This biomarker uses radiological imaging (DCE-MRIs) coupled with the biophysical simulation platform TumorScope® to predict a patient’s likelihood of pCR following treatment with immunotherapy and backbone chemotherapy. While this biomarker does not depend on transcriptomic data, it was designed by matching biological function (transcriptomics) to tumor microenvironmental features (as observed in biophysical simulations) derived from the SimBioSys TumorBank®. With this simulation-derived biomarker in hand, we validated our methods in a small, independent cohort. We additionally applied the SimbIOScope IO-prediction analysis to assess a large immunotherapy-naïve cohort, in order to validate if an increase in pCR rates in the presence of immunotherapy correlated with the anticipated rate of response to immunotherapy. Results: In TNBC tumors prior to neoadjuvant therapy, we found that a high immune evasive capability was associated with low nutrient utilization. Tumor immune evasion (including the PD-1/PDL-1 axis) is strongly correlated with tumor hypoxia (r = 0.45, p < 1 × 10^-6). Similarly, in HR+/HER2- tumors prior to neoadjuvant therapy, we found that immune evasion was negatively correlated with angiogenesis (r = -0.40, p = 0.006), suggesting that low tumor vascularization is associated with immune evasion capability. As these associations were identified from available transcriptomic data obtained from a single biopsy site within each patient’s tumor, they were unable to account for tumor heterogeneity. We therefore sought to identify a spatially-resolved biomarker for immune evasive potential in TNBC and HR+/HER2- tumors. We used publicly available DCE-MRIs of patients treated with the immunotherapy drug pembrolizumab and paclitaxel from the ISPY2 trial (n=63) to train a model to predict pCR in IO-treated tumors. Critically, the resulting model’s predictive power matched that obtained from transcriptomics data. SimbIOScope was then tested on IO-treated patients in a small, independent cohort and correctly predicted pCR in >91% (n=12). We further validated SimbIOScope by predicting the expected pCR rate in 292 patients from our virtual TumorBank in response to immunotherapy. Consistent with empirical increase (13.6% in TNBC, Keynote522) anticipated as seen from clinical trials, we found that SimbIOScope predicted a 14% increase in pCR rate in TNBC patients (and 9% increase in HR+ patients) with addition of immunotherapy versus chemotherapy backbone alone. Conclusions: The SimbIOScope platform offers physicians a rapid, non-invasive biomarker to differentiate patients likely to respond to immunotherapy from non-responders. This innovative technology thereby personalizes oncologic care and mitigates the potential for adverse effects by helping to optimize selection of patients best suited for immunotherapy. Citation Format: Gregory Norris, John Pfeiffer, Joseph Peterson, Nicole Liadis, Matthew Biancalana, Dorys Lopez-Ramos, Anuja K. Antony, Daniel Cook. Development of a Novel Imaging Biomarker to Ascertain Responsiveness to Immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-19.

Screening of GPCR drugs for repurposing in breast cancer.

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on β-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of β3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.

Abstract 5651: Characterization the mechanistic role of the proapoptotic receptor XEDAR and its therapeutic potential in TNBC

Abstract Breast cancer is one of the most common cancers and a leading cause of death in women worldwide. Among these subtypes, triple negative breast cancer (TNBC) has the worst prognosis, with a dismal outcome especially in metastatic disease. Metastatic TNBC treated with conventional chemotherapy has a median overall survival (OS) of ~ 18 months. The addition of checkpoint inhibitors extend the median OS to more than 2 years in PD-L1 positive patients, but ultimately most patients succumb to the disease. Currently TNBC still poses as a clinical unmet need, and further understanding of the cancer biology may shed light in improving therapeutics against this deadly disease. Apoptosis, or programmed cell death, is a crucial function for cell homeostasis. The ability to evade or circumvent apoptosis is an important hallmark of cancer cells. X-linked ectodermal dysplasia receptor (XEDAR), a cell membrane receptor that binds to the pro-apoptotic ligand EDA-A2, is a member of the TNF receptor family and has been reported to be downregulated in breast cancer cells. XEDAR has been shown to induce downstream apoptosis by binding to EDA-A2, by a mechanism similar to extrinsic apoptotic pathway. By interrogating clinical databases and cross comparison using TCGA, CCLE, and other breast cancer datasets, we discover XEDAR as substantially downregulated in TNBC. Transcription profile analysis between different XEDAR expressing profiles uncovered novel pathways that could be potentially targeted for TNBC treatment. RNA-seq profiling in XEDAR overexpressing TNBC cells were cross compared with aforementioned discovered pathways. Vulnerable pathways including angiogenesis were discovered as possible therapeutic targets. As a proof of concept, we combined angiogenesis inhibitors with EDA-A2 ligand treatment in XEDAR overexpression TNBC cells and observed synergistic effects for combination. Our study uncover XEDAR as a potential target for further treatment in TNBC, which remains to be further validated in preclinical and clinical studies. Citation Format: Chih-Yi Lin, Chung-Jen Yu, Chih-Yang Wang, Kai-Jun Liu, Jiun-I Lai. Characterization the mechanistic role of the proapoptotic receptor XEDAR and its therapeutic potential in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5651.

Exosomal MMP-1 transfers metastasis potential in triple-negative breast cancer through PAR1-mediated EMT.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis. Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. MMP-1 level was detected using mass spectrometry and western blot. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis. MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells, including MDA-MB-231, MDA-MB-468 and BT549. After ingesting exosomes enriched with MMP-1, cells secret more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The lung colonization model shows that the expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both upregulated. Clinically, the enrichment of MMP-1 can be detected in exosomes extracted from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of MMP-1 in exosomes is also higher than that in patients with single lesion. MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.

Endoglin Modulates TGFβR2 Induced VEGF and Proinflammatory Cytokine Axis Mediated Angiogenesis in Prolonged DEHP-Exposed Breast Cancer Cells.

Angiogenesis is the process of vascular network development and plays a crucial role in cancer growth, progression, and metastasis. Phthalates are a class of environmental pollutants that have detrimental effects on human health and are reported to increase cancer risk. However, the interplay between phthalate exposure and angiogenesis has not been investigated thoroughly. In this study, we investigated the effect of prolonged di (2-ethylhexyl) phthalate (DEHP) treatment on the angiogenic potential of triple-negative breast cancer. MDA-MB-231 cells were exposed to physiological concentrations of DEHP for more than three months. Prolonged DEHP exposure induced angiogenesis in breast cancer cells. Endoglin (ENG)/CD105 is a membrane glycoprotein and an auxiliary receptor of the TGFβ receptor complex. In endothelial cells, ENG is highly expressed and it is a prerequisite for developmental angiogenesis. A literature review highlights endoglin as a well-known mesenchymal stem cell marker responsible for vascular development and angiogenesis. NGS analysis showed that endoglin overexpression in DEHP-exposed MDA-MB-231 cells correlated with tumor development and growth. An in vivo zebrafish xenograft assay showed that VEGFA induced sprouting of the subintestinal vein (SIV) in embryos injected with DEHP-exposed cells. Endoglin knockdown reduced SIV sprouting and VEGFA expression in zebrafish embryos. An in vitro HUVEC tube formation assay showed that endoglin depletion reversed DEHP-induced VEGF-mediated HUVEC tube formation in coculture. DEHP-induced endoglin activated TGFβ/SMAD3/VEGF and MAPK/p38 signaling in MDA-MB-231 cells. A cytokine angiogenesis antibody array showed induced expression of the inflammatory cytokines IL1α, IL1β, IL6, and IL8, along with GMCSF and VEGF. Endoglin knockdown reversed DEHP-induced activation of the TGFβ/SMAD3/VEGF signaling axis, MAPK/p38 signaling, and cytokine regulation, limiting angiogenesis potential both in vivo and in vitro. Targeting endoglin might serve as a potential alternative treatment to control angiogenesis, leading to metastasis and limiting cancer progression.

Unique cartilage matrix-associated protein inhibits the migratory and invasive potential of triple-negative breast cancer

Triple-negative breast cancer (TNBC) that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is a breast cancer subtype with very aggressive metastasis and poor prognosis. Unique cartilage matrix-associated protein (UCMA) is a vitamin K-dependent protein (VKDP) with a high-density γ-carboxyglutamic acid (Gla) domain due to the action of vitamin K. UCMA promotes osteoblast differentiation and mineral deposition in bone and suppresses calcification in vessels. However, correlation between UCMA and TNBC is unknown. This study investigated the inhibitory effect of UCMA on TNBC cell in vitro migration, invasion, and colony formation in addition to in vivo tumorigenesis. Cell migration and invasion significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells compared to the mock control cells. Also, colony formation and the number of colonies significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells. These results indicate that UCMA significantly inhibits the migration, invasion, and colony formation of TNBC cells. In an in vivo xenograft mouse model, tumor growth significantly decreased in mice bearing Ucma-overexpressing TNBC cells compared to the mock control cells, indicating that UCMA reduced in vivo tumor growth, similar to the inhibitory role of UCMA in vitro. Survival analysis using publicly available database showed that high UCMA expression significantly correlated with favorable relapse-free survival in TNBC patients compared to those with the other VKDPs, matrix Gla protein (MGP) and osteocalcin (OCN). Collectively, this study suggests that UCMA is a promising new therapeutic agent for TNBC.

The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells.

There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4h (t2), and 24h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/β-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.

Arrestin Domain Containing 3 Reverses Epithelial to Mesenchymal Transition and Chemo-Resistance of TNBC Cells by Up-Regulating Expression of miR-200b.

Our previous studies demonstrated the importance of arrestin domain containing 3 (ARRDC3), a metastasis suppressor, in inhibiting invasive and metastatic potential of triple negative breast cancer (TNBC) in vitro and in vivo. However, little is known about ARRDC3 mediated transcriptional control and its target genes that are implicated in its metastatic suppressing activity. In this study, we used miRNA array and subsequent functional analyses to identify miRNAs whose expression are significantly regulated by ARRDC3 in TNBC cells. We identified miR-200b as a major target gene of ARRDC3. miR-200b played an essential role in mediating ARRDC3 dependent reversal of EMT phenotypes and chemo-resistance to DNA damaging agents in TNBC cells. Expression of miR-200b also increased the expression of ARRDC3 as well in TNBC cells, suggesting a positive feedback loop between these two molecules. In addition, we combined the therapeutic powers of miR-200b and 5-fluorourancil (5-FU) into a single compound (5-FU-miR-200b) to maximize the synergistic effects of these compounds. Chemically modified miR-200b (5-FU-miR-200b mimic) was more effective in inhibiting metastatic potentials of TNBC cells than unmodified miR-200b and does not require transfection reagents, implying its therapeutic potential in TNBC. Our studies showed the importance of therapeutic targeting ARRDC3/miR-200b pathway in TNBC.

3D-QSAR, molecular docking, and new compound design of pyrimidine derivatives as Src small molecule inhibitors

Non-receptor tyromethod, The sine kinase Src is one of the important molecular targets for potential triple-negative breast cancer (TNBC). In this study, the structure and acitivity relationship of 51 Src small molecule inhibitors with potential pyrimidine derivatives was explored. On the basis of ligand composite, the relationsConducted a phase hip between molecular structure and inhibitory activity of MDA-MB-231/435 TNBC cell lines was studied by using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis fields (CoMSIA), and Topomer CoMFA (T-COMFA) methods, and thereby two sets of three-dimensional quantitative structure–activity relationship models were established to analyze the molecular structure and anti-TNBC activity. The results indicated that the q2 of CoMFA, CoMSIA, and Topomer CoMFA models was 0.698/0.719, 0.73/0.684, 0.674/0.683, and r2 was 0.994/0.991, 0.991/0.985, 0.946/0.959, respectively. Moreover, the three-dimensional equipotential map also revealed the relationship between structural characteristics and inhibitory activity, and molecular docking was investigated by Surflex-dock. The data proved that this model had good predictive ability and can further guide the design and modification of 3- (phenylethynyl) −1 hydropyrazolyl [3anilo-d] pyrimidine-4-amine derivatives inhibitors. According to the three-dimensional quantitative structure–activity relationship model of two TNBC cell lines of MDA-MB-231/435, 13 and 11 compounds were designed respectively, and the predicted activity values showed effective inhibition of SRC.

MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors

BackgroundNon-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of yet functionally uncharacterized microRNAs in human breast carcinogenesis.MethodsIn an unbiased approach, we utilized an established model system for breast cancer (BC) stem cell formation (“mammosphere assay”) to identify whole miRNome alterations in breast carcinogenesis. Clinical samples of BC patients were used to evaluate the human relevance of the newly identified miRNA candidates. One promising candidate, miR-1287-5p, was further explored on its impact on several hallmarks of cancer. The molecular mode of action was characterized by whole transcriptome analysis, in silico prediction tools, miRNA-interaction assays, pheno-copy assays, and drug sensitivity assays.ResultsAmong several other microRNAs, miR-1287-5p was significantly downregulated in mammospheres and human BC tissue compared to normal breast tissue (p < 0.0001). Low expression levels were significantly associated with poor prognosis in BC patients. MiR-1287-5p significantly decreased cellular growth, cells in S phase of cell cycle, anchorage-independent growth, and tumor formation in vivo. In addition, we identified PIK3CB as a direct molecular interactor of miR-1287-5p and a novel prognostic factor in BC. Finally, PI3Kinase pathway chemical inhibitors combined with miR-1287-5p mimic increased the pharmacological growth inhibitory potential in triple negative BC cells.ConclusionOur data identified for the first time the involvement of miR-1287-5p in human BC and suggest a potential for therapeutic interventions in difficult to treat triple negative BC.