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Metastatic spread of tumour cells to tissues and organs around the body is the most frequent cause of death from breast cancer. This has been modelled mainly using mouse models such as syngeneic mammary cancer or human in mouse xenograft models. These have limitations for modelling human disease progression and cannot easily be used for investigation of drug resistance and novel therapy screening. To complement these approaches, advances are being made in ex vivo and 3D in vitro models, which are becoming progressively better at reliably replicating the tumour microenvironment and will in the future facilitate drug development and screening. These approaches include microfluidics, organ-on-a-chip and use of advanced biomaterials. The relevant tissues to be modelled include those that are frequent and clinically important sites of metastasis such as bone, lung, brain, liver for invasive ductal carcinomas and a distinct set of common metastatic sites for lobular breast cancer. These sites all have challenges to model due to their unique cellular compositions, structure and complexity. The models, particularly in vivo, provide key information on the intricate interactions between cancer cells and the native tissue, and will guide us in producing specific therapies that are helpful in different context of metastasis. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic and stem cell-like. Biophysical stresses such as FSS are also known to cause the production of extracellular vesicles (EVs) that can facilitate cell–cell communication by carrying biomolecular cargos such as microRNAs. Here, we hypothesized that physiological FSS will impact the yield of EV production, and that these EVs will have biomolecules that transform the recipient cells. The EVs were isolated using direct flow filtration with and without FSS from the MDA-MB-231 cancer cell line, and the expression of key stemness-related genes and microRNAs was characterized. There was a significantly increased yield of EVs under FSS. These EVs also contained significantly increased levels of miR-21, which was previously implicated to promote metastatic progression and chemotherapeutic resistance. When these EVs from FSS were introduced to MCF-7 cancer cells, the recipient cells had a significant increase in their stem-like gene expression and CD44+/CD24− cancer stem cell-like subpopulation. There was also a correlated increased proliferation along with an increased ATP production. Together, these findings indicate that the presence of physiological FSS can directly influence the EVs' production and their contents, and that the EV-mediated transfer of miR-21 can have an important role in FSS-existing contexts, such as in cancer metastasis. [ABSTRACT FROM AUTHOR]

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 23-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Background: The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. Methods and results: In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). Conclusions: These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation. [ABSTRACT FROM AUTHOR]

Recent studies have shown that stress can substantially facilitate breast cancer metastasis, which can be reduced by nonselective β1/β2-adrenergic receptor (β1/β2-AR) blocker. However, several side effects were identified. Thus, it is extremely warranted to explore more effective and better-tolerated β2-AR blocker. Currently, we demonstrated that baicalin (BA), a major bioactive component of Scutellaria baicalensis Georgi, could significantly attenuate stress hormones especially epinephrine (Epi)-induced breast cancer cell migration and invasion in vitro. Mechanistically, we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability (DARTS) combined with mass spectrum assay, and BA photoaffinity probe with pull-down assay, which was further confirmed by a couple of biophysical and biochemical assays. Furthermore, we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR, subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase (cAMP-PKA-FAK) pathway, and thus impede epithelial-mesenchymal transition (EMT), thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model. These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision‐making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one‐third of patients with ERα‐positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα‐conversion. It remains unclear whether ERα‐converted cancers are biologically similar to bona fide ERα‐negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα‐positive, ERα‐converted and ERα‐negative cases. Our data revealed that the loss of ERα‐dependency in this metastatic site leads to only a partial switch to an ERα‐negative molecular phenotype, with preservation of a luminal‐like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα‐converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα‐dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

Breast cancer is the lead cause of cancer-related deaths among women globally. Breast cancer metastasis is a complex and still inadequately understood process and a key dimension of mortality attendant to breast cancer. This study reports dysregulated genes across metastatic stages and tissues, shedding light on their molecular interplay in disease pathogenesis and new possibilities for drug discovery. Comprehensive analyses of gene expression data from primary breast tumor, circulating tumor cells, and distant metastatic sites in the brain, lung, liver, and bone were conducted. Genes dysregulated across multiple stages and tissues were identified as metastatic cascade genes, and are further classified based on functional associations with metastasis-related mechanisms. Their interactions with HUB genes in interactome networks were scrutinized, followed by pathway enrichment analysis. Validation for their potential as targets included assessments for survival, druggability, prognostic marker status, secretome annotation, protein expression, and cell type marker association. Results displayed critical genes in the metastatic cascade and those specific to metastatic sites, revealing the involvement of the collagen degradation and assembly of collagen fibrils and other multimeric structure pathways in driving metastasis. Notably, pivotal cascade genes FABP4, CXCL12, APOD, and IGF1 emerged with high metastatic potential, linked to significant druggability and survival scores, establishing them as potential molecular targets. The significance of this research lies in its potential to uncover novel biomarkers for early detection, therapeutic targets, and a deeper understanding of the molecular mechanisms underpinning the metastatic cascade in breast cancer, and with an eye to precision/personalized medicine. [ABSTRACT FROM AUTHOR]

During cancer cell invasion, integrin undergoes constant endo/exocytic trafficking. It has been found that the recycling ability of integrin β1 through Rab11-controlled long loop pathways is directly associated with cancer invasion. Previous studies showed that gain-of-function mutant p53 regulates the Rab-coupling protein [RCP]-mediated integrin β1 recycling by inactivating tumor suppressor TAp63. So, we were interested to investigate the involvement of miR-205 in this process. In the current study first, we evaluated that the lower expression of miR-205 in MDA-MB-231 cell line is associated with high motility and invasiveness. Further investigation corroborated that miR-205 directly targets RCP resulting in attenuated RCP-mediated integrin β1 recycling. Overexpression of TAp63 validates our in vitro findings. To appraise the anti-metastatic role of miR-205, we developed two in vivo experimental models- xenograft-chick embryo and xenograft-immunosuppressed BALB/c mice. Our in vivo results support the negative effect of miR-205 on metastasis. Therefore, these findings advocate the tumor suppressor activity of miR-205 in breast cancer cells and suggest that in the future development of miR-205-targeting RNAi therapeutics could be a smart alternative approach to prevent the metastatic fate of the disease. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the malignant tumors that seriously affects women’s lives and health. In recent years, the anti-tumor effect of artemisinin (ART), an active ingredient of traditional Chinese medicine, has become more prominent. However, the effectiveness of ART in the treatment of breast cancer metastasis and its mechanism are still unclear. In this paper, we used the mouse breast cancer cell 4T1 and mouse mammary epithelial cell HC11 to explore the role of ART in inhibiting metastasis and inducing apoptosis. The MTS assay showed that ART was able to significantly inhibit the viability of 4T1 cells in a concentration andtime-dependent manner. Analysis of cell colony formation, Transwell and cell scratch results revealed that ART significantly inhibited the metastasis of breast cancer 4T1 cells. Flow cytometry results showed that ART promoted ROS production, loss of mitochondrial membrane potential, cell cycle arrest and thus apoptosis in tumor cells. Bioinformatic analysis methods and studies confirmed that extremely significant high expression of TIGIT (P < 0.001) was closely associated with breast cancer metastasis, and that ART was able to significantly reduce the gene expression level of the TIGIT/CD155 signaling axis (P < 0.001, P < 0.01). In summary, this study reveals the regulatory mechanism by which ART may inhibit breast cancer metastasis by blocking the TIGIT/CD155 signaling axis, which provides a basis for ART as a potential therapeutic agent for breast cancer, and contributes to the exploration of a new way of treating tumors using the active ingredients of Chinese herbal medicine. [ABSTRACT FROM AUTHOR]

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision‐making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one‐third of patients with ERα‐positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα‐conversion. It remains unclear whether ERα‐converted cancers are biologically similar to bona fide ERα‐negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα‐positive, ERα‐converted and ERα‐negative cases. Our data revealed that the loss of ERα‐dependency in this metastatic site leads to only a partial switch to an ERα‐negative molecular phenotype, with preservation of a luminal‐like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα‐converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα‐dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients. [ABSTRACT FROM AUTHOR]

Breast cancer (BrC) is the most frequently diagnosed malignancy in woman and most BrC related deaths are due to metastasis. BrC frequently metastasizes to the lymph nodes, liver, lung, bone and brain while the urinary bladder is considered as an unusual site for breast metastasis. We report a case of bladder metastasis identified in a patient with past BrC history, presenting with hematuria, low urinary tract symptoms, and hydronephrosis.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer. [ABSTRACT FROM AUTHOR]

Aim: Method: This research presents a model combining machine learning (ML) techniques and eXplainable artificial intelligence (XAI) to predict breast cancer (BC) metastasis and reveal important genomic biomarkers in metastasis patients. Method: A total of 98 primary BC samples was analyzed, comprising 34 samples from patients who developed distant metastases within a 5-year follow-up period and 44 samples from patients who remained disease-free for at least 5 years after diagnosis. Genomic data were then subjected to biostatistical analysis, followed by the application of the elastic net feature selection method. This technique identified a restricted number of genomic biomarkers associated with BC metastasis. A light gradient boosting machine (LightGBM), categorical boosting (CatBoost), Extreme Gradient Boosting (XGBoost), Gradient Boosting Trees (GBT), and Ada boosting (AdaBoost) algorithms were utilized for prediction. To assess the models' predictive abilities, the accuracy, F1 score, precision, recall, area under the ROC curve (AUC), and Brier score were calculated as performance evaluation metrics. To promote interpretability and overcome the "black box" problem of ML models, a SHapley Additive exPlanations (SHAP) method was employed. Results: The LightGBM model outperformed other models, yielding remarkable accuracy of 96% and an AUC of 99.3%. In addition to biostatistical evaluation, in XAI-based SHAP results, increased expression levels of TSPYL5, ATP5E, CA9, NUP210, SLC37A1, ARIH1, PSMD7, UBQLN1, PRAME, and UBE2T (p ≤ 0.05) were found to be associated with an increased incidence of BC metastasis. Finally, decreased levels of expression of CACTIN, TGFB3, SCUBE2, ARL4D, OR1F1, ALDH4A1, PHF1, and CROCC (p ≤ 0.05) genes were also determined to increase the risk of metastasis in BC. Conclusion: The findings of this study may prevent disease progression and metastases and potentially improve clinical outcomes by recommending customized treatment approaches for BC patients. [ABSTRACT FROM AUTHOR]

Cancer metastasis remains the most common cause of death in breast cancer patients. Tumor‐associated macrophages (TAMs) are a novel therapeutic target for the treatment of metastatic breast cancer. Despite the good anti‐cancer activity of garcinone E (GE), there are no reports on its therapeutic effects on breast cancer metastasis. The objective of this study was to examine the anti‐cancer effects of GE on metastatic breast cancer. RAW 264.7 and THP‐1 cells were polarized to M2 macrophages by IL‐4/IL‐13 in vitro. A 4T1 mouse breast cancer model and the tail vein breast cancer metastasis model were used to explore the effect of GE on breast cancer growth and metastasis in vivo. In vitro studies showed that GE dose‐dependently suppressed IL‐4 + IL‐13‐induced expression of CD206 in both RAW 264.7 cells and differentiated THP‐1 macrophages. However, GE did not affect the LPS + IFN‐γ‐induced polarization to the M1‐like macrophages in vitro. GE inhibited the expression of the M2 macrophage specific genes in RAW 264.7 cells, and simultaneously impaired M2 macrophage‐induced breast cancer cell proliferation and migration, and angiogenesis. In animal studies, GE significantly suppressed tumor growth, angiogenesis, and lung metastasis in 4T1 tumor‐bearing mice, without causing toxicity. In both tumor and lung tissues, the proportion of M2‐like TAMs was significantly decreased while the proportion of M1‐like TAMs was markedly increased by GE treatment. Mechanistically, GE inhibited phosphorylation of STAT6 in vitro and in vivo. Our results demonstrate for the first time that GE suppresses breast cancer growth and pulmonary metastasis by modulating M2‐like macrophage polarization through the STAT6 signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Background Nitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown. Results We report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases. Conclusions S-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.

Circulating tumor cells (CTCs) are some of the key culprits that cause cancer metastasis and metastasis-related deaths. These cells exist in a dynamic microenvironment where they experience fluid shear stress (FSS), and the CTCs that survive FSS are considered to be highly metastatic and stem cell-like. Biophysical stresses such as FSS are also known to cause the production of extracellular vesicles (EVs) that can facilitate cell–cell communication by carrying biomolecular cargos such as microRNAs. Here, we hypothesized that physiological FSS will impact the yield of EV production, and that these EVs will have biomolecules that transform the recipient cells. The EVs were isolated using direct flow filtration with and without FSS from the MDA-MB-231 cancer cell line, and the expression of key stemness-related genes and microRNAs was characterized. There was a significantly increased yield of EVs under FSS. These EVs also contained significantly increased levels of miR-21, which was previously implicated to promote metastatic progression and chemotherapeutic resistance. When these EVs from FSS were introduced to MCF-7 cancer cells, the recipient cells had a significant increase in their stem-like gene expression and CD44+/CD24− cancer stem cell-like subpopulation. There was also a correlated increased proliferation along with an increased ATP production. Together, these findings indicate that the presence of physiological FSS can directly influence the EVs’ production and their contents, and that the EV-mediated transfer of miR-21 can have an important role in FSS-existing contexts, such as in cancer metastasis.

Wenli Dang,1– 3 Bin Xing,1– 3 Xintao Jia,1– 3 Ying Zhang,1– 3 Bei Jia,1– 3 Changxiang Yu,1– 3 Jiachen He,1– 3 Ziwei Li,1– 3 Huihui Li,1– 3 Zhidong Liu1– 3 1State Key Laboratory of Component-Based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China; 2Engineering Research Center of Modern Chinese Medicine Discovery and Preparation Technique, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China; 3Haihe Laboratory of Modern Chinese Medicine, Tianjin, People’s Republic of ChinaCorrespondence: Zhidong Liu, Tianjin University of Traditional Chinese Medicine, No. 10 Poyang Lake Road, 301617, Tuanbo New Town, Jinghai District, Tianjin, People’s Republic of China, Tel +86 22-59596170, Email lonerliuzd@163.comBackground: Subcellular organelle targeted nano-formulations for cancer treatment are receiving increasing attention owing to their benefits of precise drug delivery, maximized therapeutic index, and reduced off-target side effects. The nucleus and mitochondria, as the main subcellular organelles, are the significant organelles responsible for maintaining cell operation and metabolism. They can be involved in many essential physiological and pathological processes such as cell proliferation, organism metabolism, intracellular transportation, and play a critical role in regulating cell biology. Meanwhile, breast cancer metastasis is one of the leading causes of death in breast cancer patients. With the development of nanotechnology, nanomaterials have been widely used in tumor therapy.Methods: We designed a subcellular organelle targeted nanostructured lipid carriers (NLC) to deliver paclitaxel (PTX) and gambogic acid (GA) to tumor tissues.Results: Due to the surface of NLC being modified by subcellular organelle targeted peptide, the PTX and GA co-loaded NLC can accurately release PTX and GA in tumor cells. This property makes NLC able to easy to enter tumor site and target the specific subcellular organelle. The modified NLC can efficiently inhibit the growth of 4T1 primary tumor and lung metastasis, which may be related to the down-regulation of matrix metalloproteinase-9 (MMP-9) and BCL-2 levels, up-regulation of E-cadherin level, and antagonized PTX-induced increase of C-C chemokine ligand 2 (CCL-2) levels by GA. Meanwhile, the synergistic anti-tumor effect of GA and PTX has also been verified in vitro and in vivo experiments.Conclusion: The subcellular organelle targeted peptide modified PTX+GA multifunctional nano-drug delivery system has a good therapeutic effect on tumors, and this study provides significant insights into the role of different subcellular organelles in inhibiting tumor growth and metastasis and inspires researchers to develop highly effective cancer therapeutic strategies through subcellular organelle targeted drugs.Graphical Abstract: Keywords: breast cancer metastasis, gambogic acid, paclitaxel, mitochondria, nucleus

ABSTRACTThe clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response.

Background: Nitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown. Results: We report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases. Conclusions: S-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Despite the advancements in breast cancer (BrC) diagnosis and treatment, a considerable proportion of patients with early-stage disease still experience local recurrence or metastasis. This study aimed to assess the levels of specific angiogenic parameters in the EDTA plasma of BrC patients before and after treatment and to explore their clinical and prognostic significance. The levels of vascular endothelial growth factor A (VEGF-A), soluble form of vascular endothelial growth factor receptor type 1 (sVEGFR1), and soluble form of vascular endothelial growth factor receptor type 2 (sVEGFR2) were measured in 84 early BrC patients, both prior to surgery and within a median time of nine months post-treatment. Prognostic significance was evaluated using Kaplan-Meier survival and Cox regression analyses. Linear regression models were employed to examine the independent impact of selected angiogenic factors on DFS in breast cancer patients. The results of uni- and multivariate analyses indicated that a pre-treatment concentration of sVEGFR1 above 30.99 pg/mL was associated with improved disease-free survival (DFS) (p < 0.0001 for both analyses), while a pre-treatment concentration of sVEGFR2 above 9475.67 pg/mL was associated with an increased risk of BrC relapse (p < 0.0001 for both analyses). Additionally, a post-treatment concentration of sVEGFR2 above 7361.71 pg/mL was associated with better overall survival (OS) based on the Kaplan-Meier survival analysis (p = 0.0141). Furthermore, linear regression models revealed a significant inverse association between pre-treatment levels of sVEGFR1 and the risk of relapse (standardized β −0.2578, p = 0.0499) and a significant positive association of VEGF-A levels with the risk of recurrence (standardized β 0.2958, p = 0.0308). In conclusion, the findings suggest that both pre- and post-treatment levels of sVEGFR1 and sVEGFR2 may hold promise as potential prognostic markers for BrC patients. [ABSTRACT FROM AUTHOR]

Up to 30% of breast cancer (BC) patients will develop distant metastases (DM), for which there is no cure. Here, statistical and machine learning (ML) models were developed to estimate the risk of site-specific DM following local-regional therapy. This retrospective study cohort included 175 patients diagnosed with invasive BC who later developed DM. Clinicopathological information was collected for analysis. Outcome variables were the first site of metastasis (brain, bone or visceral) and the time interval (months) to developing DM. Multivariate statistical analysis and ML-based multivariable gradient boosting machines identified factors associated with these outcomes. Machine learning models predicted the site of DM, demonstrating an area under the curve of 0.74, 0.75, and 0.73 for brain, bone and visceral sites, respectively. Overall, most patients (57%) developed bone metastases, with increased odds associated with estrogen receptor (ER) positivity. Human epidermal growth factor receptor-2 (HER2) positivity and non-anthracycline chemotherapy regimens were associated with a decreased risk of bone DM, while brain metastasis was associated with ER-negativity. Furthermore, non-anthracycline chemotherapy alone was a significant predictor of visceral metastasis. Here, clinicopathologic and treatment variables used in ML prediction models predict the first site of metastasis in BC. Further validation may guide focused patient-specific surveillance practices. [ABSTRACT FROM AUTHOR]

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Metastasis is a multi-step process that leads to the dissemination of tumor cells to new sites and, consequently, to multi-organ neoplasia. Although most lethal breast cancer cases are related to metastasis occurrence, little is known about the dysregulation of each step, and clinicians still lack reliable therapeutic targets for metastasis impairment. To fill these gaps, we constructed and analyzed gene regulatory networks for each metastasis step (cell adhesion loss, epithelial-to-mesenchymal transition, and angiogenesis). Through topological analysis, we identified E2F1, EGR1, EZH2, JUN, TP63, and miR-200c-3p as general hub-regulators, FLI1 for cell-adhesion loss specifically, and TRIM28, TCF3, and miR-429 for angiogenesis. Applying the FANMOD algorithm, we identified 60 coherent feed-forward loops regulating metastasis-related genes associated with distant metastasis-free survival prediction. miR-139-5p, miR-200c-3p, miR-454-3p, and miR-1301-3p, among others, were the FFL’s mediators. The expression of the regulators and mediators was observed to impact overall survival and to go along with metastasis occurrence. Lastly, we selected 12 key regulators and observed that they are potential therapeutic targets for canonical and candidate antineoplastics and immunomodulatory drugs, like trastuzumab, goserelin, and calcitriol. Our results highlight the relevance of miRNAs in mediating feed-forward loops and regulating the expression of metastasis-related genes. Altogether, our results contribute to understanding the multi-step metastasis complexity and identifying novel therapeutic targets and drugs for breast cancer management. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that cancer-associated adipocytes (CAAs) in the tumor microenvironment are involved in the malignant progression of breast cancer. However, the underlying mechanism of CAA formation and its effects on the development of breast cancer are still unknown. Here, we show that CSF2 is highly expressed in both CAAs and breast cancer cells. CSF2 promotes inflammatory phenotypic changes of adipocytes through the Stat3 signaling pathway, leading to the secretion of multiple cytokines and proteases, particularly C–X–C motif chemokine ligand 3 (CXCL3). Adipocyte-derived CXCL3 binds to its specific receptor CXCR2 on breast cancer cells and activates the FAK pathway, enhancing the mesenchymal phenotype, migration, and invasion of breast cancer cells. In addition, a combination treatment targeting CSF2 and CXCR2 shows a synergistic inhibitory effect on adipocyte-induced lung metastasis of mouse 4T1 cells in vivo. These findings elucidate a novel mechanism of breast cancer metastasis and provide a potential therapeutic strategy for breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKC β II. Moreover, HBXIP induced the transcription of PRKCB , encoding PKC β II, by coactivating Sp1, and triggered PKC β II kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKC β II-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer. This study reveals a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and the anti-hyperlipidemic drug bezafibrate can serve as an effective anti-metastatic drug in breast cancer. [Display omitted] [ABSTRACT FROM AUTHOR]

Mediastinal metastases represent an infrequent recurrence of breast carcinoma. Esophageal involvement by metastatic tissue is extremely rare. The situation means an unusual diagnostic and therapeutic challenge. Dignity of esophageal stricture is difficult to ascertain; endoscopy and picture giving methods might be conflicting. This study aimed to analyze the clinical characteristics and the therapeutic possibilities of the condition. A retrospective analysis of malignant esophageal stricture cases was performed from a prospectively collected database between 1984 and 2020. Out of 3996 cases with esophageal malignancy, 17 esophageal strictures were confirmed to be related to breast cancer metastasis. Surgical resection was feasible in 3 cases; endoprosthesis insertion was performed for palliation in 7 cases, and in 7 cases, only supportive care was available. Our incidence rate was 0.43%. Dysphagia indicating an affected esophagus presented on average 10.5 years after the primary tumor surgery. Mean duration between onset of symptoms achieving appropriate diagnosis was 6.4 months. Dignity of the malignancy was confirmed only retrospectively in one case. Morphologic findings showed an external esophageal compression in 52.9%, while in 35.3%, a destructive tumor growth affecting the mucosal layer could be found. Overall, survival was 7.6 months referring to 15 cases. The possible occurrence of mediastinal metastases involving the esophagus should be seriously considered in patients with previous breast cancer history. Biopsies obtained from the intact mucosal surface of the stenotic esophagus are often inefficient and misleading; therefore, repeated biopsies are necessary. Options for radical surgery are highly limited. [ABSTRACT FROM AUTHOR]

The clinical successes of immune checkpoint blockade (ICB) in advanced cancer patients have recently spurred the clinical implementation of ICB in the neoadjuvant and perioperative setting. However, how neoadjuvant ICB therapy affects the systemic immune landscape and metastatic spread remains to be established. Tumors promote both local and systemic expansion of regulatory T cells (Tregs), which are key orchestrators of tumor-induced immunosuppression, contributing to immune evasion, tumor progression and metastasis. Tregs express inhibitory immune checkpoint molecules and thus may be unintended targets for ICB therapy counteracting its efficacy. Using ICB-refractory models of spontaneous primary and metastatic breast cancer that recapitulate the poor ICB response of breast cancer patients, we observed that combined anti-PD-1 and anti-CTLA-4 therapy inadvertently promotes proliferation and activation of Tregs in the tumor, tumor-draining lymph node and circulation. Also in breast cancer patients, Treg levels were elevated upon ICB. Depletion of Tregs during neoadjuvant ICB in tumor-bearing mice not only reshaped the intratumoral immune landscape into a state favorable for ICB response but also induced profound and persistent alterations in systemic immunity, characterized by elevated CD8+ T cells and NK cells and durable T cell activation that was maintained after treatment cessation. While depletion of Tregs in combination with neoadjuvant ICB did not inhibit primary tumor growth, it prolonged metastasis-related survival driven predominantly by CD8+ T cells. This study demonstrates that neoadjuvant ICB therapy of breast cancer can be empowered by simultaneous targeting of Tregs, extending metastasis-related survival, independent of a primary tumor response. [ABSTRACT FROM AUTHOR]

Aim: Method: This research presents a model combining machine learning (ML) techniques and eXplainable artificial intelligence (XAI) to predict breast cancer (BC) metastasis and reveal important genomic biomarkers in metastasis patients. Method: A total of 98 primary BC samples was analyzed, comprising 34 samples from patients who developed distant metastases within a 5-year follow-up period and 44 samples from patients who remained disease-free for at least 5 years after diagnosis. Genomic data were then subjected to biostatistical analysis, followed by the application of the elastic net feature selection method. This technique identified a restricted number of genomic biomarkers associated with BC metastasis. A light gradient boosting machine (LightGBM), categorical boosting (CatBoost), Extreme Gradient Boosting (XGBoost), Gradient Boosting Trees (GBT), and Ada boosting (AdaBoost) algorithms were utilized for prediction. To assess the models’ predictive abilities, the accuracy, F1 score, precision, recall, area under the ROC curve (AUC), and Brier score were calculated as performance evaluation metrics. To promote interpretability and overcome the “black box” problem of ML models, a SHapley Additive exPlanations (SHAP) method was employed. Results: The LightGBM model outperformed other models, yielding remarkable accuracy of 96% and an AUC of 99.3%. In addition to biostatistical evaluation, in XAI-based SHAP results, increased expression levels of TSPYL5, ATP5E, CA9, NUP210, SLC37A1, ARIH1, PSMD7, UBQLN1, PRAME, and UBE2T (p ≤ 0.05) were found to be associated with an increased incidence of BC metastasis. Finally, decreased levels of expression of CACTIN, TGFB3, SCUBE2, ARL4D, OR1F1, ALDH4A1, PHF1, and CROCC (p ≤ 0.05) genes were also determined to increase the risk of metastasis in BC. Conclusion: The findings of this study may prevent disease progression and metastases and potentially improve clinical outcomes by recommending customized treatment approaches for BC patients.

Human homolog of mouse double minute 2 (HDM2) is an oncogene frequently overexpressed in cancers with poor prognosis, but mechanisms of controlling its abundance remain elusive. In an unbiased biochemical search, we discovered Skp1-Cullin 1-FBXO22-ROC1 (SCFFBXO22) as the most dominating HDM2 E3 ubiquitin ligase from human proteome. The results of protein decay rate analysis, ubiquitination, siRNA-mediated silencing, and coimmunoprecipitation experiments support a hypothesis that FBXO22 targets cellular HDM2 for ubiquitin-dependent degradation. In human breast cancer cells, FBXO22 knockdown (KD) increased cell invasiveness, which was driven by elevated levels of HDM2. Moreover, mouse 4T1 breast tumor model studies revealed that FBXO22 KD led to a significant increase of breast tumor cell metastasis to the lung. Finally, low FBXO22 expression is correlated with worse survival and high HDM2 expression in human breast cancer. Altogether, these findings suggest that SCFFBXO22 targets HDM2 for degradation and possesses inhibitory effects against breast cancer tumor cell invasion and metastasis.

Breast cancer metastasis is an important cause of death in patients with breast cancer and is closely related to circulating tumor cells (CTCs) and the metastatic microenvironment. As the most infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs), which highly express sialic acid (SA) receptor (Siglec-1), are closely linked to tumor progression and metastasis. Furthermore, the surface of CTCs also highly expressed receptor (Selectin) for SA. A targeting ligand (SA-CH), composed of SA and cholesterol, was synthesized and modified on the surface of epirubicin (EPI)-loaded liposomes (EPI-SL) as an effective targeting delivery system. Liposomes were evaluated for characteristics, stability, in vitro release, cytotoxicity, cellular uptake, pharmacokinetics, tumor targeting, and pharmacodynamics. In vivo and in vitro experiments showed that EPI-SL enhanced EPI uptake by TAMs. In addition, cellular experiments showed that EPI-SL could also enhance the uptake of EPI by 4T1 cells, resulting in cytotoxicity second only to that of EPI solution. Pharmacodynamic experiments have shown that EPI-SL has optimal tumor inhibition with minimal toxicity, which can be ascribed to the fact that EPI-SL can deliver drugs to tumor based on TAMs and regulate TME through the depletion of TAMs. Our study demonstrated the significant potential of SA-modified liposomes in antitumor metastasis. Schematic diagram of the role of SA-CH modified EPI-loaded liposomes in the model of breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood–brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain. [ABSTRACT FROM AUTHOR]

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis. [ABSTRACT FROM AUTHOR]

Background: As per the WHO, more than 2 million new cases are diagnosed with breast cancer and more than 685,000 deaths have been reported in 2020. Incidence of recurrence and metastasis has also risen and poses a challenge for developing new therapies with the identification of newer targets. Methods: The objective of this study is to analyze and integrate various data across genomic, transcriptomic, and proteomic levels to find promising markers linked with tumor progression and metastasis development in breast cancer. This study begins with the extraction of data of differentially expressed proteins and subsequently analyzes their gene expression changes and genomic alterations to integrate all three omics data. We used curated breast cancer datasets of different proteomic experiments from dbDEPC3.0 and TCGA datasets of the Metastatic Breast Cancer Project to compare gene expressions and genomic alterations. We further used cBioportal, GeneMania, GEPIA2, Reactome, and canSAR computational tools for identifying the most significant genes associated with tumor progression and metastasis. Results: Based on false discovery rate, 14 genes were subjected to pathway analysis in Reactome and the top 25 significant pathways were analyzed out of a total of 170 pathways. Our study found fibrinogen gamma (FGG) and fibrinogen beta (FGB) linked to pathways connecting RAS-MAPK, its downstream mutants, integrin signaling, and extracellular matrix remodeling pathways. Conclusion: Survival analysis suggested that FGG (P = 0.0065) and FGB (P = 0.013) have a significant positive correlation along with their stage-wise changes in copy number variations and genomic alterations play a pivotal role in controlling tumor progression and metastasis.

HER2-positive and triple-negative breast cancers (TNBC) are difficult to treat and associated with poor prognosis. Despite showing initial response, HER2-positive breast cancers often acquire resistance to HER2-targeted therapies, and TNBC lack effective therapies. To overcome these clinical challenges, we evaluated the therapeutic utility of co-targeting TrkA and JAK2/STAT3 pathways in these breast cancer subtypes. Here, we report the novel combination of FDA-approved TrkA inhibitors (Entrectinib or Larotrectinib) and JAK2 inhibitors (Pacritinib or Ruxolitinib) synergistically inhibited in vitro growth of HER2-positive breast cancer cells and TNBC cells. The Entrectinib-Pacritinib combination inhibited the breast cancer stem cell subpopulation, reduced expression of stemness genes, SOX2 and MYC , and induced apoptosis. The Entrectinib-Pacritinib combination suppressed orthotopic growth of HER2-positive Trastuzumab-refractory breast cancer xenografts and basal patient-derived xenograft (PDXs), reduced tumoral SOX2 and MYC, and induced apoptosis in both mouse models. The Entrectinib-Pacritinib combination inhibited overall metastatic burden, and brain and bone metastases of intracardially inoculated TNBC cells without toxicity. Together, our results demonstrate for the first time that co-inhibition of TrkA and JAK2 synergistically suppresses breast cancer growth and metastasis, thereby providing preclinical evidence that supports future clinical evaluations. • TrkA and JAK2 inhibitors synergistically reduce breast cancer cell viability. • Dual targeting of TrkA and JAK2 impeded growth of breast cancer xenografts in vivo. • Co-inhibition of TrkA and JAK2 reduced metastasis burden of TNBC cells. [ABSTRACT FROM AUTHOR]

Recent studies have shown that stress can substantially facilitate breast cancer metastasis, which can be reduced by nonselective β1/β2-adrenergic receptor (β1/β2-AR) blocker. However, several side effects were identified. Thus, it is extremely warranted to explore more effective and better-tolerated β2-AR blocker. Currently, we demonstrated that baicalin (BA), a major bioactive component of Scutellaria baicalensis Georgi, could significantly attenuate stress hormones especially epinephrine (Epi)-induced breast cancer cell migration and invasion in vitro. Mechanistically, we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability (DARTS) combined with mass spectrum assay, and BA photoaffinity probe with pull-down assay, which was further confirmed by a couple of biophysical and biochemical assays. Furthermore, we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR, subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase (cAMP-PKA-FAK) pathway, and thus impede epithelial-mesenchymal transition (EMT), thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model. These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis. Our data suggest that baicalin reduces chronic stress-induced breast cancer metastasis by directly binding to and consequentially blocking the activation of β2-AR. [Display omitted] • Baicalin reduces chronic stress-induced breast cancer metastasis. • Baicalin directly binds to and consequentially blocks the activation of β2-AR. • Baicalin acts as β2-AR inhibitor to treat stress-induced breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Up to 30% of breast cancer (BC) patients will develop distant metastases (DM), for which there is no cure. Here, statistical and machine learning (ML) models were developed to estimate the risk of site-specific DM following local-regional therapy. This retrospective study cohort included 175 patients diagnosed with invasive BC who later developed DM. Clinicopathological information was collected for analysis. Outcome variables were the first site of metastasis (brain, bone or visceral) and the time interval (months) to developing DM. Multivariate statistical analysis and ML-based multivariable gradient boosting machines identified factors associated with these outcomes. Machine learning models predicted the site of DM, demonstrating an area under the curve of 0.74, 0.75, and 0.73 for brain, bone and visceral sites, respectively. Overall, most patients (57%) developed bone metastases, with increased odds associated with estrogen receptor (ER) positivity. Human epidermal growth factor receptor-2 (HER2) positivity and non-anthracycline chemotherapy regimens were associated with a decreased risk of bone DM, while brain metastasis was associated with ER-negativity. Furthermore, non-anthracycline chemotherapy alone was a significant predictor of visceral metastasis. Here, clinicopathologic and treatment variables used in ML prediction models predict the first site of metastasis in BC. Further validation may guide focused patient-specific surveillance practices.

Despite the advancements in breast cancer (BrC) diagnosis and treatment, a considerable proportion of patients with early-stage disease still experience local recurrence or metastasis. This study aimed to assess the levels of specific angiogenic parameters in the EDTA plasma of BrC patients before and after treatment and to explore their clinical and prognostic significance. The levels of vascular endothelial growth factor A (VEGF-A), soluble form of vascular endothelial growth factor receptor type 1 (sVEGFR1), and soluble form of vascular endothelial growth factor receptor type 2 (sVEGFR2) were measured in 84 early BrC patients, both prior to surgery and within a median time of nine months post-treatment. Prognostic significance was evaluated using Kaplan-Meier survival and Cox regression analyses. Linear regression models were employed to examine the independent impact of selected angiogenic factors on DFS in breast cancer patients. The results of uni- and multivariate analyses indicated that a pre-treatment concentration of sVEGFR1 above 30.99 pg/mL was associated with improved disease-free survival (DFS) (p < 0.0001 for both analyses), while a pre-treatment concentration of sVEGFR2 above 9475.67 pg/mL was associated with an increased risk of BrC relapse (p < 0.0001 for both analyses). Additionally, a post-treatment concentration of sVEGFR2 above 7361.71 pg/mL was associated with better overall survival (OS) based on the Kaplan-Meier survival analysis (p = 0.0141). Furthermore, linear regression models revealed a significant inverse association between pre-treatment levels of sVEGFR1 and the risk of relapse (standardized β −0.2578, p = 0.0499) and a significant positive association of VEGF-A levels with the risk of recurrence (standardized β 0.2958, p = 0.0308). In conclusion, the findings suggest that both pre- and post-treatment levels of sVEGFR1 and sVEGFR2 may hold promise as potential prognostic markers for BrC patients.

Breast cancer is the most commonly diagnosed cancer in the world, and metastasis is often the main cause of death in breast cancer patients. Salvia miltiorrhiza -Ginseng (SG) herb pair is clinically used for the treatment of cardiovascular diseases and cancers. However, the pharmacological action of this pair on breast cancer is yet unclear. In this study, a spontaneous metastasis model of breast cancer was constructed to assess the therapeutic value of SG. After administration of different doses of SG, the results showed that although it did not significantly inhibit tumor growth, high-dose SG administration could inhibit tumor metastasis. Then, based on systematic pharmacology combined with Gene Expression Omnibus (GEO) database, potential targets of drugs were identified such as vascular endothelial growth factor A (VEGFA), matrix metalloproteinase (MMP9), prostaglandin endoperoxide synthase2 (PTGS2), etc. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis revealed that these targets were related to cytokine-mediated signaling pathway, cell migration and other biological processes and signaling pathways such as PI3K/Akt, etc. The systematic pharmacology analysis showed that SG effectively inhibited the VEGFA and MMP9-mediated biological events such as angiogenesis, epithelial-mesenchymal transition (EMT) and impaired tumor metastasis. Overall, our research aimed to provide new ideas for the treatment of breast cancer lung metastasis in traditional Chinese medicine.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Background: Breast cancer is the most common malignancy among women worldwide. As survival rates increase, breast reconstruction and quality of life gain importance. Of all women undergoing breast reconstruction, approximately, 70% opt for silicone implants and 50% of those develop capsular contracture, the most prevalent long-term complication. The collagenase of the bacterium Clostridium histolyticum (CCH) showed promising results in the therapy of capsule contracture; however, its influence on residual cancer cells is unknown. The aim of this study was to investigate whether CCH-treatment negatively impacts breast cancer cells in vitro and in vivo. Methods: MDA-MB-231 and MCF-7 cells were used in this study. In vitro, we tested the influence of CCH on proliferation, wound healing, migration and cell cycle by MTT-assay, scratch-assay, transwell-migration-assay, and flow cytometry. In vivo, solid tumors were induced in immune-deficient mice. CCH was injected into the tumors and tumor growth and metastasis formation was monitored by caliper measurement, in vivo bioluminescence imaging and histology. Gene expression analysis was performed by microarray including 27,190 genes. Results: CCH-incubation led to a dose-dependent reduction in proliferation for both cell lines, while wound healing was reduced only in MDA-MB-231 cells. No morphological alterations were monitored in cell cycle or apoptosis. In vivo, bioluminescence imaging and histology did not show any evidence of metastasis. Although CCH led to changes in gene expression of breast cancer cells, no relevant alterations in metastasis-related genes were monitored. Conclusion: CCH has no impact on tumor growth or metastasis formation in vitro and in vivo. This paves the way for first clinical trials. [ABSTRACT FROM AUTHOR]

Host microbes are increasingly recognized as key components in various types of cancer, although their exact impact remains unclear. This study investigated the functional significance of Staphylococcus aureus (S. aureus) in breast cancer tumorigenesis and progression. We found that S. aureus invasion resulted in a compromised DNA damage response process, as evidenced by the absence of G1-phase arrest and apoptosis in breast cells in the background of double strand breaks production and the activation of the ataxia-telangiectasia mutated (ATM)-p53 signaling pathway. The high-throughput mRNA sequencing, bioinformatics analysis and pharmacological studies revealed that S. aureus facilitates breast cell metastasis through the innate immune pathway, particularly in cancer cells. During metastasis, S. aureus initially induced the expression of RIG-I-like receptors (RIG-I in normal breast cells, RIG-I and MDA5 in breast cancer cells), which in turn activated NF-κB p65 expression. We further showed that NF-κB p65 activated the CCL5-CCR5 pathway, contributing to breast cell metastasis. Our study provides novel evidence that the innate immune system, triggered by bacterial infection, plays a role in bacterial-driven cancer metastasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The migration of breast cancer cells is the main cause of death and significantly regulated by physical factors of the extracellular matrix (ECM). To be specific, the curvature and stiffness of the ECM were discovered to effectively guide cell migration in velocity and direction. However, it is not clear what the extent of effect is when these dual-physical factors regulate cell migration. Moreover, the mechanobiology mechanism of breast cancer cell migration in the molecular level and analysis of cell traction force (CTF) are also important, but there is a lack of systematic investigation. Therefore, we employed a microfluidic platform to construct hydrogel microspheres with an independently adjustable curvature and stiffness as a three-dimensional substrate for breast cancer cell migration. We found that the cell migration velocity was negatively correlated to curvature and positively correlated to stiffness. In addition, curvature was investigated to influence the focal adhesion expression as well as the assignment of F-actin at the molecular level. Further, with the help of a motor-clutch mathematical model and hydrogel microsphere stress sensors, it was concluded that cells perceived physical factors (curvature and stiffness) to cause changes in CTF, which ultimately regulated cell motility. In summary, we employed a theoretical model (motor-clutch) and experimental strategy (stress sensors) to understand the mechanism of curvature and stiffness regulating breast cancer cell motility. These results provide evidence of force driven cancer cell migration by ECM physical factors and explain the mechanism from the perspective of mechanobiology.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by α5β1integrin. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis., Competing Interests: COMPETING INTERESTS The Naba laboratory holds a sponsored research agreement with Boehringer-Ingelheim for work not related to the content of this manuscript. AN holds consulting agreements with AbbVie, XM Therapeutics, and RA Capital.

Abstract Background Metastasis represents the leading cause of death in patients with breast cancer. Traditional Chinese medicine is particularly appreciated for metastatic diseases in Asian countries due to its benefits for survival period prolongation and immune balance modulation. However, the underlying molecular mechanisms remain largely unknown. This study aimed to explore the antimetastatic effect and immunomodulatory function of a clinical formula Aiduqing (ADQ). Methods Naive CD4+ T cells, regulatory T cells (Tregs), and CD8+ T cells were sorted by flow cytometry. Then, breast cancer cells and these immune cells were co-cultured in vitro or co-injected into mice in vivo to simulate their coexistence. Flow cytometry, ELISA, qPCR, double luciferase reporter gene assay, and chromatin immunoprecipitation assay were conducted to investigate the immunomodulatory and antimetastatic mechanisms of ADQ. Results ADQ treatment by oral gavage significantly suppressed 4T1-Luc xenograft growth and lung metastasis in the orthotopic breast cancer mouse model, without noticeable hepatotoxicity, nephrotoxicity, or hematotoxicity. Meanwhile, ADQ remodeled the immunosuppressive tumor microenvironment (TME) by increasing the infiltration of tumor-infiltrating lymphocytes (TILs) and cytotoxic CD8+ T cells, and decreasing the infiltration of Tregs, naive CD4+ T cells, and tumor-associated macrophages (TAMs). Molecular mechanism studies revealed that ADQ remarkably inhibited CXCL1 expression and secretion from TAMs and thus suppressed the chemotaxis and differentiation of naive CD4+ T cells into Tregs, leading to the enhanced cytotoxic effects of CD8+ T cells. Mechanistically, TAM-derived CXCL1 promoted the differentiation of naive CD4+ T cells into Tregs by transcriptionally activating the NF-κB/FOXP3 signaling. Lastly, mouse 4T1-Luc xenograft experiments validated that ADQ formula inhibited breast cancer immune escape and lung metastasis by suppressing the TAM/CXCL1/Treg pathway. Conclusions This study not only provides preclinical evidence supporting the application of ADQ in inhibiting breast cancer metastasis but also sheds novel insights into TAM/CXCL1/NF-κB/FOXP3 signaling as a promising therapeutic target for Treg modulation and breast cancer immunotherapy. Video Abstract