Brain Metastatic Breast Cancer Cells Research Articles

DDDR-29. MATRIX STIFFNESS INDUCED PHENOTYPE IN BRAIN METASTATIC BREAST CANCER CELLS DETERMINES RESPONSE TO THERAPY

Abstract Breast cancer is the most prevalent form of cancer, representing 12.5% of all cancer cases diagnosed worldwide. Breast cancer cells can metastasize to distant organs (i.e., brain) and remain dormant for extended periods of time. This aspect makes it very difficult to treat these cells as they exhibit reduced growth as well as resistance to therapy. Despite advances in our understanding of breast cancer metastasis, the specific mechanisms that enable dormant cancer cells to resist therapeutics, particularly in brain metastatic breast cancer (BMBC), remain unclear. Herein, we employed brain tissue mimetic hyaluronic acid (HA) hydrogels of varying stiffness (i.e., ~0.4 kPa vs. ~4.5 kPa) to investigate the response to chemo or targeted therapy in dormant versus proliferative BMBC cells. It was revealed that BMBC cells grown on soft HA hydrogels (~0.4 kPa) displayed a non-proliferative (i.e., dormant) phenotype and exhibited resistance to Paclitaxel (PTX) or Lapatinib (LAP). However, BMBC cells grown on stiff HA hydrogels (~4.5 kPa) displayed a proliferative phenotype and sensitivity to PTX or LAP. Furthermore, we found that resistance to therapy was due to the enhanced expression of the serum/glucocorticoid regulated kinase 1 (SGK1) gene, mediated, in part, via the p38 mitogen-activated protein kinase (MAPK) pathway. Consequently, SGK1 inhibition using a SGK inhibitor in BMBC cells cultured on soft HA hydrogels resulted in a dormant-to-proliferative switch as well as response to PTX or LAP. In sum, our study demonstrated that matrix stiffness plays a major role in the dormancy-associated therapy response, mediated, in part, via the p38/SGK1 pathway.

TMIC-48. THREE DIMENSIONAL CANCER CELL-MONOCYTE CO-CULTURE SPHEROIDS RECAPITULATE IMMUNOSUPPRESSIVE AND PRO-TUMORIGENIC MICROENVIRONMENTS IN BRAIN METASTATIC BREAST CANCER

Abstract Brain metastasis occurs in 15 % of breast cancer patients and is one of the major causes of breast cancer related morbidity. Brain metastatic breast cancer (BMBC) microenvironment is a heterogeneous niche of cellular and acellular components of which the innate immune cells, microglia, and macrophages, comprise the major fraction. However, the role of these innate immune cells in the tumor microenvironment (TME) in mediating cancer progression and response to therapy is not well understood. To address this need, herein, we developed three dimensional (3D) co-culture spheroids using brain metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231Br or patient derived brain metastatic TNBC cell line F2-7 with THP1 monocytes. Under co-culture conditions MDA-MB-231Br co-culture spheroids assumed highly protrusive morphology with enhanced mesenchymal and proliferative characteristics. Additionally, phenotypic determination of cocultured macrophages revealed elevated levels of markers associated with alternatively activated macrophages i.e. CD163, CD206, and c-Myc. Furthermore, we found that under coculture conditions immunosuppressive and evasive markers PD1, PDL1, CD47 and CD24 were significantly upregulated. Similarly, F2-7 and monocyte cocultures also led to an immunosuppressive environment with elevated levels of M2 type markers and associated anti-inflammatory cytokines. We further found that the presence of M2 type macrophages in the microenvironment of BMBC spheroids led to acquired resistance against Paclitaxel, a clinically relevant chemotherapeutic drug against BMBC. In sum, in our coculture conditions, brain metastatic TNBC cells polarized monocytes into immunosuppressive and pro-tumorigenic tumor associated macrophages leading to a pro-tumoral microenvironment. Thus, 3D co-culture spheroids of BMBC cells and monocytes can serve as robust platform for studying cancer cell and innate immune cell interactions occurring in the BMBC environment.

Camouflaging nanoreactor traverse the blood-brain barrier to catalyze redox cascade for synergistic therapy of glioblastoma

The blood-brain barrier (BBB) is a complex and highly restrictive barrier that prevents most biomolecules and drugs from entering the brain. However, effective strategies for delivering drugs to the brain are urgently needed for the treatment of glioblastoma. Based on the efficient BBB penetration properties of exosomes derived from brain metastatic breast cancer cells (EB), this work prepared a nanoreactor (denoted as MAG@EB), which was constructed by self-assembly of Mn2+, arsenate and glucose oxidase (GOx) into nanoparticles wrapped with EB. MAG@EB can enhance the efficiency of traversing the BBB, target and accumulate at in situ glioblastoma sites. The GOx-driven glycolysis effectively cuts off the glucose supply while also providing an abundance of H2O2 and lowering pH. Meanwhile, the released Mn2+ mediated Fenton-like reaction converts elevated H2O2 into highly toxic ·OH. Besides, AsV was reduced to AsIII by glutathione, and the tumor suppressor gene P53 was activated by AsIII to kill glioblastoma cells. Glioblastoma succumbed to the redox cascade triggered by MAG@EB, as the results demonstrated in vivo and in vitro, yielding a remarkable therapeutic effect. This work provides a promising therapeutic option mediated by cascaded nanoreactors for the future treatment of glioblastoma.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also showthat brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Abstract 3157: The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis

Abstract Brain metastasis (BM) affects approximately 10% of cancer patients with metastatic disease, representing a major cause of morbidity and mortality. BM is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), particularly extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1) expression, a common immunotherapy target. Tumor suppressor p53 is shown to regulate STAT3 and ERK1/2 signaling, which are dysregulated in various malignancies. We hypothesize that p53 along with STAT3 and ERK1/2 play a critical role in the initiation and maintenance of BM. Twenty-six BM tumors with various primary sites of origin were used following IRB guidelines. pSTAT3Tyr705, pERK1/2Thr202/Tyr204, mutant p53 (p53mt), and PD-L1 expression was determined by immunohistochemistry. Gene expression analysis of BM was performed on a cDNA microarray. Human brain metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 inhibitor (NSC74859) or MAPK inhibitor (U0126) in regular or astrocytic media to determine cell proliferation and migration by MTT assay and scratch-wound, respectively. ERK1/2 signaling pathway status was determined by Western blotting. Results demonstrated: 1) pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed profusely at tumor margins, while p53mt was uniformly expressed throughout. 81% of BM expressed p53mt while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. Venn analysis displayed significant overlap between expression, with 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between both pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt. Expression of PD-L1 was seen in a majority of BM tumors. 2) Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes; these included upregulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. 3) Cell proliferation showed augmented growth in astrocytic media (p≤0.05) and was significantly decreased following treatment with NSC74850 or U0126 (p≤0.01). While migration showed a trend towards significance in reduction following NSC74859 (p=0.08), migration stagnated following U0126 treatment in astrocytic media (p≤0.01). 4) pERK1/2Thr202/Tyr204 expression was reduced following U0126 or NSC74859 treatment. These findings suggest activation of STAT3 and ERK1/2 promote BM and suppression of these pathways may lead to a reduction in cell viability and motility. Therefore, this provides compelling evidence for the use of STAT3 and ERK1/2 along with p53 as potential targets for immunotherapy in brain metastatic disease. Citation Format: Sabrina L. Zeller, Eris Spirollari, John V. Wainwright, Simon J. Hanft, Chirag D. Gandhi, Meena Jhanwar-Uniyal. The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis [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 3157.

O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

Matrix stiffness influences response to chemo and targeted therapy in brain metastatic breast cancer cells.

Breast cancer is the most common malignancy accounting for 12.5% of all newly diagnosed cancer cases across the globe. Breast cancer cells are known to metastasize to distant organs (i.e., brain), wherein they can exhibit a dormant phenotype for extended time periods. These dormant cancer cells exhibit reduced proliferation and therapeutic resistance. However, the mechanisms by which dormant cancer cells exhibit resistance to therapy, in the context of brain metastatic breast cancer (BMBC), is not well understood. Herein, we utilized hyaluronic acid (HA) hydrogels with varying stiffnesses to study drug responsiveness in dormant vs. proliferative BMBC cells. It was found that cells cultured on soft HA hydrogels (∼0.4 kPa) that showed a non-proliferative (dormant) phenotype exhibited resistance to Paclitaxel or Lapatinib. In contrast, cells cultured on stiff HA hydrogels (∼4.5 kPa) that showed a proliferative phenotype exhibited responsiveness to Paclitaxel or Lapatinib. Moreover, dormancy-associated resistance was found to be due to upregulation of the serum/glucocorticoid regulated kinase 1 (SGK1) gene which was mediated, in part, by the p38 signaling pathway. Accordingly, SGK1 inhibition resulted in a dormant-to-proliferative switch and response to therapy. Overall, our study demonstrates that matrix stiffness influences dormancy-associated therapy response mediated, in part, via the p38/SGK1 axis.

Brain Metastases Are Regulated by Immuno-inflammatory Signaling Pathways Governed by STAT3, MAPK and Tumor Suppressor p53 Status: Possible Therapeutic Targets.

Brain metastasis (BM) is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1), an immunotherapy target. Tumor suppressor p53, dysregulated in cancers, regulates STAT3 and ERK1/2 signaling. This study examined the roles of STAT3, MAPK and p53 status in BM initiation and maintenance. Twenty-six BM, with various primary malignancies, were used (IRB-approved) to determine mutant p53 (p53mt), pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 expression using immunohistochemistry. cDNA microarray was used for gene expression analysis. Brain-metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 (NSC74859) or MAPK/ERK1/2 (U0126) inhibitors in regular or astrocytic media. ERK1/2 pathway was assessed using western blotting, and cell proliferation and migration were determined using MTT and scratch-wound assays, respectively. pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed at tumor margins, whereas p53mt and PD-L1 were uniformly expressed, with significant overlap between expression of these proteins. Gene expression analysis identified alterations in 18 p53- and 32 STAT3- or MAPK-associated genes contributing to dysregulated immune responses and cell cycle regulation. U0126 and NSC74859 reduced pERK1/2Thr202/Tyr204 expression. Cell proliferation decreased following each treatment (p≤0.01). Migration stagnated following U0126 treatment in astrocytic media (p≤0.01). Activation of STAT3 and ERK1/2 promotes BM and provides compelling evidence for use of STAT3, ERK1/2 and p53 status as potential immunotherapeutic targets in BM.

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

Microglia increase CEMIP expression and promote brain metastasis in breast cancer through the JAK2/STAT3 signaling pathway

Abstract Objectives Brain metastasis is the most lethal metastatic site for patients with breast cancer, and the incidence of brain metastasis is increasing every year. Microglia act a pivotal part in promoting the proliferation and metastasis of breast cancer cells in the brain. Therefore, understanding the biological process of brain metastasis in breast cancer is important to improve therapeutic outcomes and prolong the survival of patients. Materials and Methods The role of microglia on the prognosis of patients with breast cancer with brain metastasis was verified by immunohistochemistry and the Kaplan–Meier curve. Cell experiments in vitro were used to analyze the effect of microglia on cell proliferation, migration and invasion. Knockdown of cell migration-inducing hyaluronan-binding protein (CEMIP) expression and co-culture experiments were carried out to study the mechanism of microglia on the progression of brain metastasis of breast cancer. Results We found that microglia may shorten the survival time of patients with breast cancer by regulating the expression of CEMIP in brain metastatic tumors. Co-culture experiments in vitro indicated that microglia enhance the proliferation, migration, and invasion abilities of brain metastatic breast cancer cells; however, the knockdown of CEMIP expression suppresses this effect. In addition, we also found that CEMIP expression, increased by microglia, activates the JAK2/STAT3 pathway in brain metastatic breast cancer cells, which induces the secretion of CCL2, IL-6, TGF-β, and VEGF. CCL2 recruits microglia to gather around brain metastases, whereas IL-6, TGF-β, and VEGF induce high CEMIP expression, triggering a positive feedback loop between microglia and brain metastatic breast cancer cells. Conclusions Our study proposes a possible mechanism of microglia promoting brain metastasis of breast cancer, indicating that both microglia and CEMIP may be valuable therapeutic targets for patients with breast cancer with brain metastasis.

CNSC-02. GLUTAMATERGIC SYNAPTIC INPUT DRIVES BRAIN METASTASIS OF BREAST CANCER AND MELANOMA

Abstract The nervous system is implicated in pathology of both cranial and extracranial cancers. In glioblastoma, direct excitatory glutamatergic synapses between neurons and cancer cells were discovered. Importantly, these neuron-cancer contacts drive glioblastoma proliferation and invasion. However, such synapses have not yet been documented in other types of brain cancer. In the context of brain metastases, indirect, peri-synaptic contacts between breast cancer cells and neurons were observed, which promote brain metastatic growth through glutamatergic signaling via NMDA receptors on the neoplastic cells. Recently, two studies have demonstrated that neuronal activity supports the growth of both the primary tumor and brain metastases from neuroendocrine carcinoma small cell lung cancer. In the work presented here, we demonstrate direct neuron-cancer synapses in early-stage brain metastases originating from breast cancer and melanoma. These synapses exhibited the typical structural features observed in synaptic connections and induced postsynaptic currents in the cancer cells through the activation of AMPA subtype glutamate receptors. Importantly, genetic and pharmacological perturbation of these receptors reduces the number of brain metastases and the overall metastatic burden in preclinical models of breast cancer and melanoma. These results uncover a previously unknown form of direct communication between neurons and brain-metastatic breast cancer and melanoma tumor cells. This neuronal-tumor synaptic interaction demonstrates the significance of the neuronal microenvironment in the early stages of the brain metastatic cascade. Furthermore, it unveils a potential therapeutic vulnerability associated with the neuronal microenvironment, suggesting that targeting the synaptic communication between neurons and cancer cells could be a promising strategy to impede the progression of brain metastases.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early‐Stage Glioma

AbstractGlioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early‐stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non‐targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug‐loaded nanoparticles, the as‐obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early‐stage glioma.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early-Stage Glioma.

Glioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early-stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non-targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug-loaded nanoparticles, the as-obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early-stage glioma.

DH5α Outer Membrane-Coated Biomimetic Nanocapsules Deliver Drugs to Brain Metastases but not Normal Brain Cells via Targeting GRP94.

Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.

MiR-623 Targets Metalloproteinase-1 and Attenuates Extravasation of Brain Metastatic Triple-Negative Breast Cancer Cells.

BackgroundMost breast cancer-related deaths result from metastasis. Understanding the molecular basis of metastasis is needed for the development of effective targeted and preventive strategies. Matrix metalloproteinase-1 (MMP1) plays an important role in brain metastasis (BM) of triple-negative breast cancer (TNBC) by promoting extravasation of cancer cells across the brain endothelium (BE). MMP1 expression is controlled by endogenous microRNAs. Preliminary bioinformatics analysis has revealed that miR-623, known to target the 3ʹUTR of MMP1, is significantly downregulated in brain metastatic tumors compared to primary BC tumors. However, the involvement of miR-623 in MMP1 upregulation in breast cancer brain metastatic cells (BCBMC) remains unexplored. Here, we investigated the role of miR-623 in MMP1 regulation and its impact on the extravasation of TNBC cells through the BE in vitro.Materials and MethodsA loss-and-gain of function method was employed to address the effect of miR-623 modulation on MMP1 expression. MMP1 regulation by miR-623 was investigated by real-time PCR, western blot, luciferase and transwell migration assays using an in vitro human BE model.ResultsOur results confirmed that brain metastatic TNBC cells express lower levels of miR-623 compared with cells having low propensity to spread toward the brain. miR-623 binds to the 3′-untranslated region of MMP1 transcript and downregulates its expression. Restoring miR-623 expression significantly decreased MMP1 expression, preserved the endothelial barrier integrity, and attenuated transmigration of BCBMC through the BE.ConclusionOur study elucidates, for the first time, the crucial role of miR-623 as MMP1 direct regulator in BCBMC and sheds light on miR-623 as a novel therapeutic target that can be exploited to predict and prevent brain metastasis in TNBC. Importantly, the presents study helps in unraveling a brain metastasis-specific microRNA signature in TNBC that can be used as a guide to personalized metastasis prediction and preventive approach with better therapeutic outcome.

OGT/CDK5/ACSS2 Axis Regulates Breast Cancer Brain Metastasis Growth

Onset of brain metastases (BMs) in breast cancer patients is considered an end‐stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Thus, there is an urgent need to develop novel treatment strategies. Metastatic breast cancer cells colonizing the brain encounter adverse ‘nutritional environment’, as the levels of key metabolic fuels such as glucose are much lower, in part because they are avidly consumed by neurons. To overcome this challenge, brain‐metastatic cancer cells rely on acetate as a fuel source and convert it into acetyl‐CoA via upregulation of acetyl‐CoA synthetase 2 (ACSS2) enzyme. We have previously shown that brain tumors increase the nutrient sensing post‐translational modification O‐GlcNAcylation and O‐GlcNAc transferase (OGT) levels to regulate acetate metabolism into acetyl‐CoA via increased phosphorylation of ACSS2 on Ser‐267 in a cyclin dependent kinase 5 (CDK5)‐dependent manner, increasing brain tumor growth. Here, we show that human breast cancer cells selected to metastasize to the brain contain increased levels of O‐GlcNAc, OGT and ACSS2‐Ser267 phosphorylation compared to parental cells and show that human breast cancer brain metastatic patient samples contain elevated ACSS2‐Ser‐267 levels. Moreover, overexpression of OGT or ACSS2‐S267D phospho‐mimetic mutant confer a growth advantage on brain metastatic breast cancer cells in an in vivo intracranial xenograft model, may be due to an increase in lipogenic proteins. Additionally, we show that pharmacologically targeting CDK5 and ACSS2 with small molecule inhibitors reduces tumor growth in a novel orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling in transducing nutritional state to regulate acetate metabolism in breast cancer BM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastasis.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.

Paclitaxel (PTX) is a first-line chemotherapeutic drug for breast cancer, but PTX resistance often occurs in metastatic breast cancer. In addition, due to the poor targeting of chemotherapeutic drugs and the presence of the blood-brain barrier (BBB), it is hard to effectively treat brain metastatic breast cancer using paclitaxel. Thus, it is urgent to develop an effective drug delivery system for the treatment of brain metastatic breast cancer. The current study found that TWF1 gene, an epithelial-mesenchymal transition-associated gene, was overexpressed in brain metastatic breast cancer (231-BR) cells and was associated with the PTX resistance of 231-BR cells. Knockdown of TWF1 by small interference RNA (siRNA) in 231-BR cells could effectively increase the sensitivity of brain metastatic breast cancer cells to paclitaxel. Then, a liposome-based drug delivery system was developed for PTX delivery across BBB, enhancing PTX sensitivity and brain metastases targeting via BRBP1 peptide modification. The results showed that BRBP1-modified liposomes could effectively cross the BBB, specifically accumulate in brain metastases, and effectively interfere TWF1 gene expression in vitro and in vivo, and thus they enhanced proliferation inhibition, cell cycle arrest, and apoptosis induction, thereby inhibiting the formation and growth of brain metastases. In summary, our results indicated that BRBP1-modified and PTX- and TWF1 siRNA-loaded liposomes have the potential for the treatment of brain metastatic breast cancer, which lays the foundation for the development of a new targeted drug delivery system.

Comparing the fate of brain metastatic breast cancer cells in different immune compromised mice with cellular magnetic resonance imaging.

Metastasis is the leading cause of mortality in breast cancer patients, with brain metastases becoming increasingly prevalent. Studying this disease is challenging due to the limited experimental models and methods available. Here, we used iron-based cellular MRI to track the fate of a mammary carcinoma cell line (MDA-MB-231-BR) in vivo to characterize the growth of brain metastases in the nude and severely immune-compromised NOD/SCID/ILIIrg-/- (NSG) mouse. Nude and NSG mice received injections of iron-labeled MDA-MB-231-BR cells. Images were acquired with a 3T MR system and assessed for signal voids and metastases. The percentage of signal voids and the number and volume of metastases were quantified. Ex vivo imaging of the liver, histology, and immunofluorescence labeling was performed. Brain metastases grew more rapidly in NSG mice. At day 21 post cell injection, the average number of brain tumors in NSG mice was approximately four times greater than in nude mice. The persistence of iron-labeled cells, visualized as signal voids by MRI, was also examined. The percentage of voids decreased significantly over time for both nude and NSG mice. Body images revealed that the NSG mice also had metastases in the liver, lungs, and lymph nodes while tumors were only detected in the brains of nude mice. This work demonstrates the advantages of using the highly immune-compromised NSG mouse to study breast cancer metastasis, treatments aimed at inhibiting metastasis and outgrowth of breast cancer metastases in multiple organs, and the role that imaging can play toward credentialing these models that cannot be done with other in vitro or histopathologic methods alone.