Regulation Of Breast Cancer Metastasis Research Articles

Abstract A034: Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis

Abstract Triple-negative breast cancer (TNBC) is associated with a high risk of distant metastasis, particularly to the lung and liver. Both intrinsic characteristics of cancer cells and the tumor microenvironment (TME) influence TNBC growth and metastasis. The TME, comprising stromal, immune, and endothelial cells, exhibits heterogeneity both between and within cell types. Identifying the molecular characteristics of TME cells that affect cancer progression is crucial. We aim to investigate this using single-cell RNA sequencing of patient-derived xenograft (PDX) models to explore TME cells and genes involved in TNBC metastasis. We established PDX models by transplanting tumor tissues from 26 TNBC patients into immunodeficient mice. These models were categorized based on their metastatic potential and patient outcomes: non-metastatic (n=5) and metastatic (n=4). Single-cell RNA sequencing of nine PDX tumors revealed significant differences in gene expression of murine stromal cells between metastatic and non-metastatic models. We identified ten genes (Serpinb2, Spp1, Tnc, Thbs1, Timp1, Il11, Mt2, Crabp1, Cck, Mt1) that were highly expressed in the stromal cells of metastatic PDX models. To test if TNBC cells influence these fibroblast genes, we exposed NIH3T3 fibroblasts to conditioned media from the 4T1 TNBC cell line. In three independent experiments, only Crabp1 expression in NIH3T3 cells was consistently increased by 4T1- conditioned media. CRABP1, a retinoic acid-binding protein, is known for its role in differentiation and proliferation by regulating MAPK signaling. Although Crabp1-positive cancer-associated fibroblasts have been noted, its role in TNBC metastasis was previously unexplored. We investigated CRABP1 function using a CRABP1 knockdown NIH3T3 cell line. CRABP1 knockdown led to reduced invasion and migration of 4T1 cells in trans-well assays and decreased invasiveness of 4T1 spheroids in a collagen matrix. Additionally, CRABP1 knockdown NIH3T3 cells showed reduced proliferation in vitro and fewer alpha-SMA positive cancer-associated fibroblasts in co-injected 4T1 tumors in BALB/C mice. In summary, CRABP1, identified through single-cell RNA sequencing of stromal cells in TNBC PDX models, is a potential regulator of TNBC metastasis, affecting cancer cell migration, invasion, and fibroblast proliferation. Citation Format: Woohang Heo, Yujeong Her, Sieun Yang, Dakyung Lee, Rokhyun Kim, Jong-Il Kim, Hyeong-Gon Moon. Single cell RNA sequencing of triple negative breast cancer patient-derived xenograft model identifies CRABP1 of cancer-associated fibroblast as a key regulator of breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A034.

Tumor-associated macrophages induce epithelial-mesenchymal transition and promote lung metastasis in breast cancer by activating the IL-6/STAT3/TGM2 axis

Breast cancer is one of the most common tumors in the world and metastasis is the major cause of tumor-related death. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and often associated with cancer metastasis. Nevertheless, the mechanism by which TAMs regulate breast cancer metastasis remain unclear. In this study, we found that transglutaminase 2 (TGM2) could serve as a crucial target in the modulation of TAMs-induced epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells. Further analysis revealed that IL-6 secreted from TAMs, which was capable of inducing TGM2 expression through the activation of the JAK/STAT3 signaling pathway. Subsequent luciferase reporter assays demonstrated that STAT3 binds to the TGM2 promoter region, thereby transcriptionally enhancing TGM2 expression. In conclusion, our current research has identified the IL-6/STAT3/TGM2 axis as a pivotal regulator in breast tumorigenesis caused by TAMs, presenting a novel target for the treatment of breast cancer.

Molecular Mechanism of lncRNAs in Regulation of Breast Cancer Metastasis; a Comprehensive Review.

Although the number of breast cancer deaths has decreased, and there have been developments in targeted therapies and combination treatments for the management of metastatic illness, metastatic breast cancer is still the second most common cause of cancer-related deaths in U.S. women. Numerous phases and a vast number of proteins and signaling molecules are involved in the invasion-metastasis cascade. The tumor cells penetrate and enter the blood or lymphatic vessels, and travel to distant organs via the lymphatic or blood vessels. Tumor cells enter cell cycle arrest, adhere to capillary beds in the target organ, and then disseminate throughout the organ's parenchyma, proliferating and enhancing angiogenesis. Each of these processes is regulated by changes in the expression of different genes, in which lncRNAs play a role in this regulation. Transcripts that are longer than 200 nucleotides and do not translate into proteins are called RNAs. LncRNA molecules, whose function depends on their unique molecular structure, play significant roles in controlling the expression of genes at various epigenetic levels, transcription, and so on. LncRNAs have essential functions in regulating the expression of genes linked to cell development in healthy and pathological processes, specialization, programmed cell death, cell division, invasion, DNA damage, and spread to other parts of the body. A number of cancer types have been shown to exhibit aberrant expression of lncRNAs. In this review, we describe the general characteristics, potential molecular mechanisms and targeted therapy of lncRNAs and discuss the emerging functions of lncRNAs in breast cancer.

Abstract 1776 Nuclear APLF in the regulation of breast cancer metastasis

Abstract 1776 Nuclear APLF in the regulation of breast cancer metastasis

Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

Redox signalling regulates breast cancer metastasis via phenotypic and metabolic reprogramming due to p63 activation by HIF1α.

Redox signaling caused by knockdown (KD) of Glutathione Peroxidase 2 (GPx2) in the PyMT mammary tumour model promotes metastasis via phenotypic and metabolic reprogramming. However, the tumour cell subpopulations and transcriptional regulators governing these processes remained unknown. We used single-cell transcriptomics to decipher the tumour cell subpopulations stimulated by GPx2 KD in the PyMT mammary tumour and paired pulmonary metastases. We analyzed the EMT spectrum across the various tumour cell clusters using pseudotime trajectory analysis and elucidated the transcriptional and metabolic regulation of the hybrid EMT state. Integration of single-cell transcriptomics between the PyMT/GPx2 KD primary tumour and paired lung metastases unraveled a basal/mesenchymal-like cluster and several luminal-like clusters spanning an EMT spectrum. Interestingly, the luminal clusters at the primary tumour gained mesenchymal gene expression, resulting in epithelial/mesenchymal subpopulations fueled by oxidative phosphorylation (OXPHOS) and glycolysis. By contrast, at distant metastasis, the basal/mesenchymal-like cluster gained luminal and mesenchymal gene expression, resulting in a hybrid subpopulation using OXPHOS, supporting adaptive plasticity. Furthermore, p63 was dramatically upregulated in all hybrid clusters, implying a role in regulating partial EMT and MET at primary and distant sites, respectively. Importantly, these effects were reversed by HIF1α loss or GPx2 gain of function, resulting in metastasis suppression. Collectively, these results underscored a dramatic effect of redox signaling on p63 activation by HIF1α, underlying phenotypic and metabolic plasticity leading to mammary tumour metastasis.

Search for Novel Antagonist/S of HIF-1α from Selected Synthetic Analgesics/ Bioactive Flavonoids: An In-Silico Approach

Computer aided drug designing as well as drug repurposing implies the usage of molecular modelling techniques like analysis of the structures of receptor and ligand, molecular docking, pharmacokinetics and toxicity prediction, to explain the bioactivity of the synthetic molecules or plant secondary metabolites to design more efficient drug candidates or to repurpose an old drug in new diseases. Numerous studies have demonstrated that the low oxygen environment inside the cell is a key factor in developing breast cancer metastasis. To gain insight into the spread of breast cancer, hypoxia-inducible factor 1 (HIF-1), one of the master regulators of the hypoxic response, has been intensively explored. Our current research focuses on the insilico analysis and comparative study to evaluate the effects of different cancer drugs, analgesics, and plant-derived flavonoid compounds on HIF-1α regulation of breast cancer metastasis. According to the study, Quercetin shows the maximum binding affinity, i.e., -8.2 kcal/ mol. followed by Letrozole (-7.3 kcal/mol.), Naringenin (-7.11 kcal/mol), Tamoxifen (-7.07 kcal/mol), Phenacetin (-6.16 kcal/mol), and Aspirin (-5.7 kcal/mol). The study highlighted that Quercetin has the strongest binding affinity whereas Aspirin has the least binding affinity with HIF-1α protein. Hence the least toxic compound Quercetin can be a good candidate to control breast cancer metastasis by modulating the HIF-1 pathway.

Epigenetic regulation of breast cancer metastasis.

Breast cancer is the most frequently diagnosed malignancy and the second leading cause of cancer-related mortality among women worldwide. Recurrent metastasis is associated with poor patient outcomes and poses a significant challenge in breast cancer therapies. Cancer cells adapting to a new tissue microenvironment is the key event in distant metastasis development, where the disseminating tumor cells are likely to acquire genetic and epigenetic alterations during the process of metastatic colonization. Despite several decades of research in this field, the exact mechanisms governing metastasis are not fully understood. However, emerging body of evidence indicates that in addition to genetic changes, epigenetic reprogramming of cancer cells and the metastatic niche are paramount toward successful metastasis. Here, we review and discuss the latest knowledge about the salient attributes of metastasis and epigenetic regulation in breast cancer and crucial research domains that need further investigation.

Mass cytometry analysis of mouse nonclassical monocyte heterogeneity in breast tumors

Abstract Monocytes have emerged as important regulators of cancer progression. Our previous studies demonstrated the importance of nonclassical Ly6C lomonocytes in regulating cancer metastasis in mouse models. Here, we aimed to study monocyte heterogeneity in two different breast cancer tumors in mice using either 4T1 or EMT6 cells. 4T1 tumors exhibit an increased ability to metastasize in the lung in a BALB/c mouse breast cancer model compared to the less aggressive EMT6 tumor model. We evaluated 35 surface markers of tumor-infiltrated monocytes and dendritic cells in tumors isolated from each model using CyTOF mass cytometry. Analysis of monocytes and dendritic cells in the tumors using the Seurat package and the FlowSom algorithm identified 12 cell subsets. Significant differences were discovered between the two tumor types in frequencies of four of 12 identified subsets. However, only one of these subsets, a nonclassical monocyte subset, was higher in the less metastatic EMT6 tumors. This subset was 5-fold higher in EMT6 tumors versus more aggressive 4T1 tumors. This Ly6C lononclassical monocyte subset showed high expression of CD169 and CX3CR1, CD86 and CD80. These differentially expressed markers suggest a functional importance in this subset in migration to the tumor and antigen presentation, both of which are anti-tumoral functions. Ongoing studies are aimed towards understanding the role of this non-classical monocyte population in regulating breast cancer metastasis. This work was supported by NIH R01 CA202987, and P01 HL136275

Altered tRNA Isoacceptor Abundance Regulates Breast Cancer Metastasis.

Two tRNA isoleucyl isoacceptors have opposing effects on metastatic colonization in breast cancer.

Circadian-rhythm-regulating hormones: Key factors to regulate breast cancer metastasis via circulating tumor cells.

Circadian-rhythm-regulating hormones: Key factors to regulate breast cancer metastasis via circulating tumor cells.

A yes-associated protein 1- Notch1 receptor positive feedback loop promotes breast cancer lung metastasis by attenuating the bone morphogenetic protein 4-SMAD family member 1/5 signaling.

The Notch1 (Notch1 receptor) and yes-associated protein 1 (YAP1) signaling can regulate breast cancer metastasis. This study aimed at investigating whether and how these two signal pathways crosstalk to promote breast cancer lung metastasis. Here, we show that YAP1 expression was positively correlated with Notch1 in breast cancer according to bioinformatics and experimental validation. Mechanistically, YAP1 with TEA domain transcription factors (TEADs) enhanced Jagged1(JAG1)-Notch1 signaling. Meanwhile, Notch1 promoted YAP1 stability in breast cancer cells by inhibiting the β-TrCP-mediated degradation, thereby, forming a YAP1- JAG1/Notch1 positive feedback loop in breast cancer. Furthermore, YAP1 enhanced the mammosphere formation and stemness of MDA-MB-231 cells by attenuating the inhibition of the BMP4-SMAD1/5 signaling. In vivo, the YAP1- JAG1/Notch1 positive feedback loop promoted the lung colonization of MDA-MB-231 cells. Our data for the first time indicate that the YAP1-Notch1 positive feedback loop promotes lung metastasis of breast cancer by modulating self-renewal and inhibiting the BMP4-SMAD1/5 signaling.

CDDO, a PPAR-γ ligand, inhibits TPA-induced cell migration and invasion through a PPAR-γ-independent mechanism.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) acts as a key factor in breast cancer metastasis. Notably, PPAR-γ can inhibit metalloproteinase (MMP), which is involved in cancer metastasis. Our previous study revealed that PPAR-γ was related to breast cancer metastasis. The present study aimed to investigate whether the PPAR-γ ligand 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) mediated suppression of cell invasion and reduced the expression of MMP-9 in breast cancer cells. The results indicated that CDDO reduced MMP-9 expression, cell migration and invasion of breast cancer cells by inhibiting TPA-induced phosphorylation of mitogen-activated protein kinases, and downregulating the activities of activator protein-1 and nuclear factor κB. Notably, knock-out of PPAR-γ by small interfering RNA in MCF-7 cells revealed that TPA-induced MMP-9 expression occurred through a PPAR-γ-independent pathway. These data indicated that the downregulatory effect of CDDO on MMP-9 expression was affected by a mechanism independent of PPAR-γ. In conclusion, the findings of the present study suggested that CDDO may act as a key agent in the regulation of breast cancer metastasis, suggesting CDDO as a new targeted therapy for breast cancer.

NUPR1 promotes the proliferation and migration of breast cancer cells by activating TFE3 transcription to induce autophagy

Recurrence and metastasis affect the survival rate of breast cancer patients. The fundamental reason lies in the lack of understanding of the mechanism of breast cancer metastasis. In this study, the proliferation, migration and invasion abilities of breast cancer cells were evaluated. The mechanism of NUPR1/TFE3 signaling pathway on autophagy-related proteins and migration-invasion-related proteins was examined in cell model in vitro. The effects of NUPR1 on malignancy formation and metastasis were investigated in vivo. We found that NUPR1 was upregulated in breast cancer cells and tissues. NUPR1 knockdown inhibited the proliferation, migration and invasion of ZR-75-30 cells and inhibited malignancy formation and metastasis in vivo. Mechanically, NUPR1 promoted autophagy by activating of TFE3 transcription, thereby regulating breast cancer metastasis. This paper indicates that NUPR1 activates autophagy through the TFE3 signaling pathway to promote breast cancer metastasis, and provides a biological basis for the intervention of blocking distant metastasis.

Transcription factor Runx2 changes the expression of some matricellular proteins in metastatic breast cancer cells.

Runx2 is one of the runt-related genes that are overexpressed in human cancers and contribute to metastasis. The cancer cell metastasis requires modifications of the extracellular matrix (ECM) and reduction in ECM-cell interaction. This process is performed by various enzymes and proteins secreted by cancer and surrounding cells. This study aimed to investigate the effect of the Runx2 transcription factor on the expression of matricellular proteins such as HPA1, LOX, SPARC, and OPN, which have important roles in ECM modification and ECM-cell interaction in human breast cancer. Also, the changes in their associated oncogenic pathways including Akt, Erk, FAK activities, and c-jun protein expression were investigated. Runx2 knockdown model was created using runx2 siRNA in MDA-MB-231 human metastatic breast cancer cells. The changes in the mRNA and protein expressions of ECM proteins were shown by the qPCR and Western blotting, respectively. The results showed that there was a decrease in both mRNA and protein expressions of HPA1, SPARC, and LOX, whereas there was no change in those of OPN. Phosphorylated Akt, Erk, FAK levels, and protein expression of c-jun, however, decreased in the cells. Our results revealed that Runx2 affected matricellular protein expression, which is important for metastasis and invasion of breast cancer. Hence, we have concluded that runx2 appears to be efficient for regulating breast cancer metastasis through an expression of matricellular proteins.

Integrative multiomics analysis identifies a metastasis-related gene signature and the potential oncogenic role of EZR in breast cancer.

Distant metastasis is a major cause of increased mortality in breast cancer patients, but the mechanisms underlying breast cancer metastasis remain poorly understood. In this study, we aimed to identify a metastasis-related gene (MRG) signature for predicting progression in breast cancer. By screening using three regression analysis methods, a 9-gene signature (NOTCH1, PTP4A3, MMP13, MACC1, EZR, NEDD9, PIK3CA, F2RL1 and CCR7) was constructed based on an MRG set in the BRCA cohort from TCGA. This signature exhibited strong robustness, and its generalizability was verified in the Metabric and GEO cohorts. Of the nine MRGs, EZR is an oncogenic gene with a well-documented role in cell adhesion and cell migration, but it has rarely been investigated in breast cancer. Based on a search of different databases, EZR was found to be significantly more highly expressed in both breast cancer cells and breast cancer tissue. EZR knockdown significantly inhibited cell proliferation, invasion, chemoresistance and EMT in breast cancer. Mechanistically, RhoA activation assays confirmed that EZR knockdown inhibited the activity of RhoA, Rac1 and Cdc42. In summary, we identified a nine-MRG signature that can be used as an efficient prognostic indicator for breast cancer patients, and owing to its involvement in regulating breast cancer metastasis, EZR might serve as a therapeutic target.

The Jumonji Domain-Containing Histone Demethylase Homolog 1D/lysine Demethylase 7A (JHDM1D/KDM7A) Is an Epigenetic Activator of RHOJ Transcription in Breast Cancer Cells.

The small GTPase RHOJ is a key regulator of breast cancer metastasis by promoting cell migration and invasion. The prometastatic stimulus TGF-β activates RHOJ transcription via megakaryocytic leukemia 1 (MKL1). The underlying epigenetic mechanism is not clear. Here, we report that MKL1 deficiency led to disrupted assembly of the RNA polymerase II preinitiation complex on the RHOJ promoter in breast cancer cells. This could be partially explained by histone H3K9/H3K27 methylation status. Further analysis confirmed that the H3K9/H3K27 dual demethylase JHDM1D/KDM7A was essential for TGF-β-induced RHOJ transcription in breast cancer cells. MKL1 interacted with and recruited KDM7A to the RHOJ promoter to cooperatively activate RHOJ transcription. KDM7A knockdown attenuated migration and invasion of breast cancer cells in vitro and mitigated the growth and metastasis of breast cancer cells in nude mice. KDM7A expression level, either singularly or in combination with that of RHOJ, could be used to predict prognosis in breast cancer patients. Of interest, KDM7A appeared to be a direct transcriptional target of TGF-β signaling. A SMAD2/SMAD4 complex bound to the KDM7A promoter and mediated TGF-β-induced KDM7A transcription. In conclusion, our data unveil a novel epigenetic mechanism whereby TGF-β regulates the transcription of the prometastatic small GTPase RHOJ. Screening for small-molecule inhibitors of KDM7A may yield effective therapeutic solutions to treat malignant breast cancers.

Biomechanical regulation of breast cancer metastasis and progression

Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3Kγ inhibitor.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Long non-coding RNAs in breast cancer metastasis.

Breast cancer is the leading cause of cancer-related death among women. Recurrence of primary tumor and metastasis to distant body parts are major causes of breast cancer-associated mortality. The 5-year survival rate for women with metastatic breast cancer is only 25–30%. Breast cancer metastasis is a series of processes involved with EMT, invasion, loss of cell to cell adhesion, alteration in cell phenotype, extravasation, microenvironment of the tumor, and colonization to the secondary sites. Epigenetic modification is involved in the transformation of the distant stromal cell into a secondary tumor. LncRNAs, are one the key epigenetic modifiers, are the largest endogenous non-coding RNAs with approximate base-pair lengths from 200 nt to 100 kb. LncRNA plays a crucial role in breast cancer metastasis by sponging miRNA, by degrading or silencing specific mRNA, or else by targeting the enzymes and microprocessor subunits involved in the biogenesis of miRNA. LncRNA also alters the expression of several genes involved in breast cancer metastasis and modulating different cell signaling pathways. The goal of this review is to provide a better understanding of the role of lncRNA in the regulation of breast cancer metastasis. We also summarized some of the key lncRNAs that regulate the genes and signaling pathways involved in breast cancer invasion and metastasis.