Triple-negative Breast Cancer Migration Research Articles

The effect of ubiquitin ligase Cullin3 on the proliferation, migration and invasion of triple-negative breast cancer cells and exploration of its mechanism

Objective: To investigate the effects of ubiquitin ligase Cullin3 (CUL3) on the proliferation, migration and invasion ability of triple-negative breast cancer (TNBC) cells and its mechanism of action. Methods: Bioinformatics-based methods were used to obtain CUL3 gene and protein expression data in TNBC tissues, and to assess the expression of CUL3 in tumour tissues of TNBC patients (n=160) and in normal breast tissues (n=572), and its relationship with clinical prognosis. The effects of overexpression of CUL3 on the proliferation, migration and invasion ability of TNBC cells in vitro were detected by CCK8 cell proliferation assay, scratch assay and transwell assay; proteins that might interact with CUL3 were screened by immunoprecipitation combined with mass spectrometry analysis, and the substrate protein regulated by CUL3 was identified as Glutathione S-Transferase Pi 1 (GSTP1); the effects of overexpression of GSTP1 on the migration and invasion ability of TNBC cells were detected by scratch assay and Transwell assay, and it was explored whether overexpression of CUL3 could reverse the effects of GSTP1 on the migration and invasion ability of cells; and the effects of overexpression of GSTP1 on the migration and invasion ability of cells were detected by Western blot and IP (Immunoprecipitation) to detect the effect of CUL3 on the ubiquitination modification of GSTP1 protein, and to verify the molecular mechanism by which CUL3 regulates the expression of GSTP1 to affect TNBC migration and invasion. Results: CUL3 expression was significantly higher in TNBC (P<0.000 1), and high CUL3 expression was closely associated with poor prognosis of TNBC patients (OS, P=0.018; RFS, P=0.008); overexpression of CUL3 significantly increased the proliferation of TNBC cells (F=11.97, P=0.002 for the 231-cell group, F=51.92, P<0.001 for the 468-cell group), migration [74.7±4.0 and 128.0±6.1 perforating cells in the overexpression groups of 231 and 468 cell lines, compared with 21.0±2.7 and 70.0±6.6 in the blank control (NC) group, and the t-values of 231 and 468 cell groups were-19.24 and-11.23, with P-values<0.001] and invasive ability (48 h cell proliferation rates were 56.6%±4.4% and 51.6%±3.7% in the 231 and 468 cell line overexpression groups, compared with 40.5%±2.9% and 32.9%±4.8% in the NC group, respectively, t=-5.26, P=0.006 3 in the 231 cell group; t=-5.38 in the 468 cell group, P=0.005 8); GSTP1 expression was reduced in TNBC, and up-regulation of GSTP1 inhibited TNBC cell migration (the number of membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines were 16.3±6.5 and 33.0±6.2, respectively, compared with 34.3±2.5 and 77.3±5.0 in the NC group, and t=5.44 in the 231 cell group, P=0.006; 468 cell group t=7.20, P=0.002) and invasion (48 h cell proliferation rates of 49.6%±1.7% and 36.2%±1.4% in the 231 and 468 cell line overexpression groups, compared to 59.4%±4.7% and 53.0%±1.7% in the NC group, t=3.42, P=0.027 in the 231 cell group; 468 cell group t=13.18, P<0.001), whereas up-regulation of CUL3 reversed the effects of GSTP1 on cell migration (37.0±1.0 and 67.0±5.3 membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines, respectively, 231 cell group t=-3.97, P=0.017; 468 cell group t=-6.12, P=0.004), and invasion (48 h cell proliferation rates of 71.9%±3.6% and 59.4%±2.1% in the 231 and 468 cell line overexpression groups, respectively, with t-values of -9.61 and -16.01 in the 231 and 468 cell groups, respectively, P-values<0.001) inhibitory effects; and CUL3, by increasing GSTP1 ubiquitylation modification, promotes ubiquitin-proteasome system to degrade GSTP1 protein, thereby reducing the stability of GSTP1 protein. Conclusion: Overexpression of CUL3 promotes TNBC development by promoting GSTP1 ubiquitination degradation inducing cell migration and invasion.

Extracellular matrix markerLAMC2 targets ZEB1 to promote TNBC malignancy via up-regulating CD44/STAT3 signaling pathway.

Triple negative breast cancer (TNBC) is a heterogeneous and aggressive disease characterized by a high risk of mortality and poor prognosis. It has been reported that Laminin γ2 (LAMC2) is highly expressed in a variety of tumors, and its high expression is correlated with cancer development and progression. However, the function and mechanism by which LAMC2 influences TNBC remain unclear. Kaplan-Meier survival analysis and Immunohistochemical (IHC) staining were used to examine the expression level of LAMC2 in TNBC. Subsequently, cell viability assay, wound healing and transwell assay were performed to detect the function of LAMC2 in cell proliferation and migration. A xenograft mouse model was used to assess tumorigenic function of LAMC2 in vivo. Luciferase reporter assay and western blot were performed to unravel the underlying mechanism. In this study, we found that higher expression of LAMC2 significantly correlated with poor survival in the TNBC cohort. Functional characterization showed that LAMC2 promoted cell proliferation and migration capacity of TNBC cell lines via up-regulating CD44. Moreover, LAMC2 exerted oncogenic roles in TNBC through modulating the expression of epithelial-mesenchymal transition (EMT) markers. Luciferasereporterassay verified that LAMC2 targeted ZEB1to promote its transcription.Interestingly, LAMC2 regulated cell migration in TNBC via STAT3 signaling pathway. LAMC2 targeted ZEB1 via activating CD44/STAT3 signaling pathway to promote TNBC proliferation and migration, suggesting that LAMC2 could be a potential therapeutic target in TNBC patients.

Structural clarification of mannoglucan GSBP-2 from Ganoderma sinense and its effects on triple-negative breast cancer migration and invasion

Ganoderma sinense, known as Lingzhi in China, is a medicinal fungus with anti-tumor properties. Herein, crude polysaccharides (GSB) extracted from G. sinense fruiting bodies were used to selectively inhibit triple-negative breast cancer (TNBC) cells. GSBP-2 was purified from GSB, with a molecular weight of 11.5 kDa and a composition of α-l-Fucp-(1→, β-d-Glcp-(1→, β-d-GlcpA-(1→, →3)-β-d-Glcp-(1→, →3)-β-d-GlcpA-(1→, →4)-α-d-Galp-(1→,→6)-β-d-Manp-(1→, and →3,6)-β-d-Glcp-(1→ at a ratio of 1.0:6.3:1.7:5.5:1.5:4.3:8.0:7.9. The anti-MDA-MB-231 cell activity of GSBP-2 was determined by methyl thiazolyl tetrazolium, colony formation, scratch wound healing, and transwell migration assays. The results showed that GSBP-2 could selectively inhibit the proliferation, migration, and invasion of MDA-MB-231 cells through the regulation of genes targeting epithelial–mesenchymal transition (i.e., Snail1, ZEB1, VIM, CDH1, CDH2, and MMP9) in the MDA-MB-231 cells. Furthermore, Western blotting results indicated that GSBP-2 could restrict epithelial–mesenchymal transition by increasing E-cadherin and decreasing N-cadherin expression through the PI3K/Akt pathway. GSBP-2 also suppressed the angiogenesis of human umbilical vein endothelial cells. In conclusion, GSBP-2 could inhibit the proliferation, migration, and invasion of MDA-MB-231 cells and showed significant anti-angiogenic ability. These findings indicate that GSBP-2 is a promising therapeutic adjuvant for TNBC.

Gradient Rotating Magnetic Fields Impairing F-Actin-Related Gene CCDC150 to Inhibit Triple-Negative Breast Cancer Metastasis by Inactivating TGF-β1/SMAD3 Signaling Pathway.

Triple-negative breast cancer (TNBC) is the most aggressive and lethal malignancy in women, with a lack of effective targeted drugs and treatment techniques. Gradient rotating magnetic field (RMF) is a new technology used in oncology physiotherapy, showing promising clinical applications due to its satisfactory biosafety and the abundant mechanical force stimuli it provides. However, its antitumor effects and underlying molecular mechanisms are not yet clear. We designed two sets of gradient RMF devices for cell culture and animal handling. Gradient RMF exposure had a notable impact on the F-actin arrangement of MDA-MB-231, BT-549, and MDA-MB-468 cells, inhibiting cell migration and invasion. A potential cytoskeleton F-actin-associated gene, CCDC150, was found to be enriched in clinical TNBC tumors and cells. CCDC150 negatively correlated with the overall survival rate of TNBC patients. CCDC150 promoted TNBC migration and invasion via activation of the transforming growth factor β1 (TGF-β1)/SMAD3 signaling pathway invitro and invivo. CCDC150 was also identified as a magnetic field response gene, and it was marked down-regulated after gradient RMF exposure. CCDC150 silencing and gradient RMF exposure both suppressed TNBC tumor growth and liver metastasis. Therefore, gradient RMF exposure may be an effective TNBC treatment, and CCDC150 may emerge as a potential target for TNBC therapy.

Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and leads to the poorest patient outcomes despite surgery and chemotherapy treatment. Exploring new molecular mechanisms of TNBC that could lead to the development of novel molecular targets are critically important for improving therapeutic options for treating TNBC.MethodsWe sought to identify novel therapeutic targets in TNBC by combining genomic and functional studies with lipidomic analysis, which included mechanistic studies to elucidate the pathways that tie lipid profile to critical cancer cell properties. Our studies were performed in a large panel of human breast cancer cell lines and patient samples.ResultsComprehensive lipid profiling revealed that phospholipid metabolism is reprogrammed in TNBC cells. We discovered that patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is overexpressed in TNBC cell lines and tissues from breast cancer patients. Silencing of PNPLA8 disrupted phospholipid metabolic reprogramming in TNBC, particularly affecting the levels of phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and glycerophosphocholine (GPC). We showed that PNPLA8 is essential in regulating cell viability, migration and antioxidation in TNBC cells and promoted arachidonic acid and eicosanoid production, which in turn activated PI3K/Akt/Gsk3β and MAPK signaling.ConclusionsOur study highlights PNPLA8 as key regulator of phospholipid metabolic reprogramming and malignant phenotypes in TNBC, which could be further developed as a novel molecular treatment target.

Inhibition of CD36 and Nogo-B expression inhibited the proliferation and migration of triple negative breast cancer cells

Cluster of differentiation 36 (CD36) is a membrane glycoprotein receptor capable of binding and transporting fatty acid. Nogo-B regulates the metabolism of fatty acids in the liver and affects the development of liver cancer. To date, it remains unclear whether the interaction between CD36 and Nogo-B affects the proliferation and migration of breast cancer cells. In the current study, we aimed to determine whether the interference of CD36 and Nogo-B affects the proliferation and migration of triple-negative breast cancer (TNBC) cells. The results showed that inhibition of CD36 or Nogo-B alone can inhibit the proliferation and migration of TNBC cells, and the inhibitory effect was more pronounced when CD36 and Nogo-B were inhibited simultaneously. Meanwhile, it was found that inhibition of CD36 and Nogo-B expression can inhibit the expression of Vimentin, B-cell lympoma-2 (BCL2) and proliferating cell nuclear antigen (PCNA). In vivo, knockdown of CD36 or Nogo-B in E0771 cells reduced its tumorigenic ability, which was further enhanced by knockdown of CD36 and Nogo-B simultaneously. Mechanistically, inhibition of CD36 and Nogo-B expression can decrease fatty acid binding protein 4 (FABP4) and fatty acid transport protein 4 (FATP4) expression. Moreover, overexpression of CD36 and Nogo-B-induced cell proliferation was attenuated by FABP4 siRNA, indicating that inhibition of CD36 and Nogo-B expression could inhibit the absorption and transport of fatty acids, thereby inhibiting the proliferation and migration of TNBC. Furthermore, inhibition of CD36 and Nogo-B expression activated the P53-P21-Rb signaling pathway which contributed to the CD36 and Nogo-B-inhibited proliferation and migration of TNBC. Taken together, the results suggest that inhibition of CD36 and Nogo-B can reduce the proliferation and migration of TNBC, which provides new targets for the development of drugs against TNBC.

Scutellarin suppresses the metastasis of triple-negative breast cancer via targeting TNFα/TNFR2-RUNX1-triggered G-CSF expression in endothelial cells

Triple-negative breast cancer (TNBC) is heterogeneous and aggressive, with high vascularity and frequent metastasis. We have already found natural flavonoid scutellarin (SC) suppressed spontaneous TNBC metastasis via normalizing tumor vasculature in vivo. In this study, supernatant from tumor necrosis factorα (TNFα)-treated human mammary microvascular endothelial cell (HMMEC) promoted cell migration and pseudopod formation in TNBC cells, but these phenomena were disappeared in SC-co-treated HMMEC. TNFα enhanced the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF) in both HMMEC and human umbilical vein endothelial cell (HUVEC). G-CSF promoted TNBC migration and invasion in vitro, while G-CSF neutralization antibody and SC both inhibited TNBC metastasis in Balb/c mice. SC had no inhibition on the G-CSF-induced TNBC cell migration, but reduced G-CSF content in TNBC tumor tissues and TNFα-stimulated endothelial cells (ECs). SC restricted the nuclear translocation of runt-related transcription factor 1 (RUNX1) in TNBC tumor vessels and TNFα-treated ECs. RUNX1 was found to directly bind to the promoter of G-CSF in TNBC tumor vessels and regulated G-CSF expression. TNF receptor 2 (TNFR2) was crucial for regulating the TNFα-induced RUNX1 activation and G-CSF expression. Notably, SC hindered the interaction between TNFα and TNFR2 via binding to TNFR2. This work demonstrated that SC reduced TNBC metastasis by targeting TNFα/TNFR2-initiated RUNX1 activation and subsequent G-CSF production in TNBC-associated ECs.

PRMT5 triggers glucocorticoid-induced cell migration in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are the most aggressive breast cancers, and therapeutic options mainly rely on chemotherapy and immunotherapy. Although synthetic glucocorticoids (GCs) are given to alleviate the side effects of these treatments, GCs and their receptor, the glucocorticoid receptor (GR), were recently associated with detrimental effects, albeit the mechanisms involved remain elusive. Here, we identified the arginine methyltransferase PRMT5 as a master coregulator of GR, serving as a scaffold protein to recruit phospho-HP1γ and subsequently RNA polymerase II, independently of its methyltransferase activity. Moreover, the GR/PRMT5/HP1γ complex regulated the transcription of GC-target genes involved in cell motility and triggering cell migration of human TNBC cells in vitro and in a zebrafish model. Of note, we observed that GR/PRMT5 interaction was low in primary tumors but significantly increased in residual tumors treated with chemotherapy and GCs in neoadjuvant setting. These data suggest that the routine premedication prescription of GCs for early TNBC patients should be further assessed and that this complex could potentially be modulated to specifically target deleterious GR effects.

A novel transcription factor SIPA1: identification and verification in triple-negative breast cancer

Transcription factors (TFs) regulate the expression of genes responsible for cell growth, differentiation, and responses to environmental factors. In this study, we demonstrated that signal-induced proliferation-associated 1 (SIPA1), known as a Rap-GTPase-activating protein, bound DNA and served as a TF. Importin β1 was found to interact with SIPA1 upon fibronectin treatment. A TGAGTCAB motif was recognized and bound by DNA-binding region (DBR) of SIPA1, which was confirmed by electrophoretic mobility shift assay. SIPA1 regulated the transcription of multiple genes responsible for signal transduction, DNA synthesis, cell adhesion, cell migration, and so on. Transcription of fibronectin 1, which is crucial for cell junction and migration of triple-negative breast cancer (TNBC) cells, was regulated by SIPA1 in a DBR-dependent manner both in vivo and in vitro. Furthermore, single-cell transcriptome sequencing analysis of specimens from a metastatic TNBC patient revealed that SIPA1 was highly expressed in metastatic TNBC. Hence, this study demonstrated that SIPA1 served as a TF, promoting TNBC migration, invasion, and metastasis.

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK-1 activity of triple-negative breast cancers by reducing the glycolysis.

Among all the subtypes of breast cancer, triple-negative breast cancer (TNBC) has been associated with the worst prognosis. Recently, for many solid tumors (including breast cancer) metabolic reprogramming has appeared as a cancer cell hallmark, and the elevated glycolytic pathway has been linked to their aggressive phenotype. In the present study, we evaluated the prognostic and therapeutic relevance of PFKFB3 (6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase) in TNBCs. Prognostic significance of PFKFB3 expression was evaluated in overall breast cancers as well as in TNBCs. PFKFB3 inhibitor (3PO potent analogue i.e., PFK15) cytotoxicity in TNBC cell lines (MDA-MB-231 and MDA-MB-468) was analyzed using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cancer cell physiological characteristics like clonogenicity and migration were also investigated after PFK15 treatment. As fructose-2,6-bisphosphate (F-2,6-BP), has been associated with increased PFK-1 activity, the effect of PFKFB3 inhibition by PFK15 was investigated on two major isoforms of phosphofructokinase-1 (PFK-1) in breast cancer, that is, phosphofructokinase-platelet type (PFKP) and phosphofructokinase-liver type (PFKL) (relevant to breast cancer). For PFKL inhibition, the siRNA approach was used. PFKFB3 expression was significantly correlated with inferior overall survival in breast cancer patients including TNBCs. PFK15 treatment in TNBC cells (i.e., MDA-MB-231 and MDA-MB-468) resulted in a decreased PFKP expression, thereby leading to reduced colony formation ability, migration rate, and extracellular lactate levels. However, to our surprise PFK15 treatment in both TNBC cells also resulted in elevated PFKL levels. Our results demonstrated that the combinatorial inhibition of PFK15 with siPFKL was more effective in TNBC cells, as it led to a decrease in colony formation ability, migration rate, extracellular lactate levels, and PFK-1 activity when compared with individual treatments. Using bona fide PFKFB3 inhibitor, that is, AZ67, we further show that AZ67 treatment to TNBC cells has no effect either on the expression of PFKP and PFKL, or on the lactate production. In summary, our present in vitro study demonstrated that 3PO derived PFK15 mechanism of action is totally different from AZ67 in TNBC cells. However, we advocate that the PFK15-mediated inhibition (along with PFKL) on the TNBCs migration, colony formation, and PFK-1 activity can be further explored for the therapeutic advantage of TNBC patients.

EGFR-Targeted Liposomes Combined with Ginsenoside Rh2 Inhibit Triple-Negative Breast Cancer Growth and Metastasis.

Triple-negative breast cancer (TNBC) remains the most challenging breast cancer subtype due to its lack of targeted therapies and poor prognosis. In order to treat patients with these tumors, efforts have been made to explore feasible targets. Epidermal growth factor receptor (EGFR)-targeted therapy is currently in clinical trials and regarded to be a promising treatment strategy. In this study, an EGFR-targeting nanoliposome (LTL@Rh2@Lipo-GE11) using ginsenoside Rh2 as a wall material was developed, in which GE11 was used as the EGFR-binding peptide to deliver more ginsenoside Rh2 and luteolin into TNBC. In comparison to non-targeted liposomes (Rh2@Lipo and LTL@Rh2@Lipo), the nanoliposomes LTL@Rh2@Lipo-GE11 demonstrated a high specificity to MDA-MB-231 cells that expressed a high level of EGFR both in vitro and in vivo, contributing to the strong inhibitory effects on the growth and migration of TNBC. These results suggest that LTL@Rh2@Lipo-GE11 is a prospective candidate for targeted therapy of TNBC, with a remarkable capability to inhibit tumor development and metastasis.

Enhanced mitochondrial fission inhibits triple-negative breast cancer cell migration through an ROS-dependent mechanism

Mitochondria produce reactive oxygen species (ROS), which function in signal transduction. Mitochondrial dynamics, encompassing morphological shifts between fission and fusion, can directly impact ROS levels in cancer cells. In this study, we identified an ROS-dependent mechanism for how enhanced mitochondrial fission inhibits triple negative breast cancer (TNBC) cell migration. We found that enforcing mitochondrial fission in TNBC resulted in an increase in intracellular ROS levels and reduced cell migration and the formation of actin-rich migratory structures. Consistent with mitochondrial fission, increasing ROS levels in cells inhibited cell migration. Conversely, reducing ROS levels with either a global ormitochondrially targeted scavenger overcame the inhibitory effects of mitochondrial fission. Mechanistically, we found that the ROS sensitive SHP-1/2 phosphatases partially regulate inhibitory effects of mitochondrial fission on TNBC migration. Overall, our work reveals the inhibitory effects of ROS in TNBC and supports mitochondrial dynamics as a potential therapeutic target for cancer.

Exploring the mechanism of an active ingredient of ginger, dihydrocapsaicin, on triple negative breast cancer based on network pharmacology and invitro experiments.

To investigate the potential mechanism of ginger in the treatment of triple-negative breast cancer (TNBC) based on network pharmacology, molecular docking and in vitro cell experiments. The Traditional Chinese Medicine Systems Pharmacology Database And Analysis Platform, the Bioinformatics Analysis Tool For Molecular Mechanism Of Traditional Chinese Medicine and the HERB database and literature search were used to search for the main active compounds of ginger. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were used to predict the possible molecular mechanism and signaling pathway of ginger in the treatment of triple negative breast cancer. The key core genes of ginger in the treatment of triple negative breast cancer were docked with the active ingredients of ginger on the Autodock platform, and the mechanism of ginger on triple negative breast cancer was further verified by in vitro cell experiments. As a result, 10 effective components, 27 potential targets and 10 Protein-Protein Interaction core genes were predicted in the treatment of triple negative breast cancer with ginger, involving 287 biological processes, 18 cellular components and 38 molecular functions. Ginger regulated the proliferation, migration and apoptosis of triple negative breast cancer cells by regulating TNF, IL-17, FoxO, MAPK, PI3K/AKT and other signaling pathways. The results of molecular docking showed that the lowest binding potential energy between dihydrocapsaicin (DHC) and EGFR protein was -7.70 kcal·mol-1, followed by that between 6-gingerol and EGFR protein was -7.30 kcal·mol-1 and that between DHC and CASP3 protein was -7.20 kcal·mol-1. In vitro cell experiments showed that ginger could inhibit the proliferation and migration of TNBC MDA-MB-231 cells, increase the mRNA expression of Caspase family CASP9 and the protein expression of CASP3 and BAX. Overall, based on the combination of network pharmacology and in vitro cell experiments, ginger has the characteristics of multi-target in the treatment of TNBC, which may play a regulatory role through the PI3K/AKT family. It provides a reference for the drug development of ginger and the clinical treatment of triple negative breast cancer.

Knockdown of NCAPG promotes the apoptosis and inhibits the invasion and migration of triple‑negative breast cancer MDA‑MB‑231 cells via regulation of EGFR/JAK/STAT3 signaling.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and the treatment options are extremely limited. Non-SMC condensing I complex subunit G (NCAPG) expression is upregulated in TNBC, but its specific regulatory mechanism in TNBC has not been previously reported. The expression levels of NCAPG in TNBC were analyzed using data obtained from the UALCAN database. RT-qPCR and western blotting were used to detect the expression of NCAPG in various breast cancer cell lines. The expression of NCAPG was knocked down, and cell viability was then detected using a CCK-8 assay, apoptosis was detected using a TUNEL assay, and the expression of the apoptosis-related proteins Bcl-2, Bax and Bad were detected by western blotting. Wound healing and Transwell assays were used to assess migration and invasion. Western blotting was also used to determine the expression levels of migration and invasion-related proteins MMP2 and MMP9, as well as EGFR/JAK/STAT3 pathway-related proteins. Following exogenous treatment with EGF and the JAK/STAT3 signaling pathway agonist colivelin, cell viability, apoptosis, invasion and migration were assessed. The expression of NCAPG in TNBC MDA-MB-231 cells was significantly increased. Inhibition of NCAPG inhibited the activity, promoted apoptosis, and inhibited the invasion and migration of TNBC MDA-MB-231 cells, potentially via regulation of the EGFR/JAK/STAT3 signaling pathway. In conclusion, downregulation of NCAPG can promote apoptosis and inhibit invasion and migration of TNBC MDA-MB-231 cells via EGFR/JAK/STAT3 signaling.

Z-Guggulsterone Induces Cell Cycle Arrest and Apoptosis by Targeting the p53/CCNB1/PLK1 Pathway in Triple-Negative Breast Cancer.

Myrrh is the dried resin of Commiphora Myrrh Engl., which exerts anticancer properties. However, its effects and molecular mechanisms in triple-negative breast cancer (TNBC) remain unclear. In this study, we used network pharmacology to screen Z-Guggulsterone (Z-GS) as a characteristic active component of myrrh. Cell Counting Kit-8 proliferation assays showed that Z-GS inhibited proliferation of the TNBC cell lines MDA-MB-468 and BT-549. Transwell assays also showed that Z-GS inhibited TNBC migration and invasion phenotypes. Our network pharmacology combined with RNA-sequencing analyses showed that Z-GS affected cell cycle and apoptosis processes in TNBC cells, mainly via p53 signaling, to regulate key CCNB1 (cyclin B1), PLK1 (polo-like kinase 1), and p53 targets. Flow cytometry revealed that Z-GS arrested the cell cycle at the G2/M phase and increased apoptosis in TNBC cells. Western blotting and quantitative real-time polymerase chain reaction studies confirmed that Z-GS functioned via the p53-mediated downregulation of CCNB1 and PLK1 expression. In vivo studies showed that Z-GS effectively inhibited TNBC progression. Collectively, Z-GS exhibited potential anti-TNBC activity and may functions via the p53/CCNB1/PLK1 pathway.

The synthetic molecule stauprimide impairs cell growth and migration in triple-negative breast cancer

Stauprimide, a semi-synthetic derivative of staurosporine, is known mainly for its potent differentiation-enhancing properties in embryonic stem cells. Here, we studied the effects of stauprimide in cell growth and migration of triple-negative breast cancer cells in vitro, evaluating its potential antitumoral activity in an orthotopic mouse model of breast cancer in vivo. Our results from survival curves, EdU incorporation, cell cycle analysis and annexin-V detection in MDA-MB-231 cells indicated that stauprimide inhibited cell proliferation, arresting cell cycle in G2/M without induction of apoptosis. A decrease in the migratory capability of MDA-MB-231 was also assessed in response to stauprimide. In this work we pointed to a mechanism of action of stauprimide involving the modulation of ERK1/2, Akt and p38 MAPK signalling pathways, and the downregulation of MYC in MDA-MB-231 cells. In addition, orthotopic MDA-MB-231 xenograft and 4T1 syngeneic models suggested an effect of stauprimide in vivo, increasing the necrotic core of tumors and reducing metastasis in lung and liver of mice. Together, our results point to the promising role of stauprimide as a putative therapeutic agent in triple-negative breast cancer.

EZH2-H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis

Triple-Negative Breast Cancer (TNBC) has a poor prognosis and adverse clinical outcomes among all breast cancer subtypes as there is no available targeted therapy. Overexpression of Enhancer of zeste homolog 2 (EZH2) has been shown to correlate with TNBC’s poor prognosis, but the contribution of EZH2 catalytic (H3K27me3) versus non-catalytic EZH2 (NC-EZH2) function in TNBC progression remains elusive. We reveal that selective hyper-activation of functional EZH2 (H3K27me3) over NC-EZH2 alters TNBC metastatic landscape and fosters its peritoneal metastasis, particularly splenic. Instead of H3K27me3-mediated repression of gene expression; here, it promotes KRT14 transcription by attenuating binding of repressor SP1 to its promoter. Further, KRT14 loss significantly reduces TNBC migration, invasion, and peritoneal metastasis. Consistently, human TNBC metastasis displays positive correlation between H3K27me3 and KRT14 levels. Finally, EZH2 knockdown or H3K27me3 inhibition by EPZ6438 reduces TNBC peritoneal metastasis. Altogether, our preclinical findings suggest a rationale for targeting TNBC with EZH2 inhibitors.

Macrophage CCL22 Secretion Under Hypoxic Conditions Promotes the Metastasis of Triple-Negative Breast Cancer

Objective: To explore the mechanism by which macrophages secrete CCL22 to promote the metastasis of triple-negative breast cancer under hypoxic conditions. Methods: 20 ng/mL mass concentration of phorbol 12-myristate 13-acetate (PMA) cell culture medium, 4?,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), trypsin digestion solution, CCL18 Kit , Interleukin (IL)-10 Kit, CCL17 Kit, CCL22 Kit, TRIzolTM Reagent Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Kit, triple-negative breast cancer cells MDA-MB-231 and BT-549, as well as other reagents were used to culture triple-negative breast cancer (TNBC) cells MDA-MB-231 and BT-549 as well as human mononuclear cells THP-1, analyze and observe the metastasis of triple-negative breast cancer cells to the lungs, the secretion of CCL22, the migration of triple-negative breast cancer, and the situation of CCR4. Results: Compared with normal tumor-associated macrophage (TAM), hypoxic TAM further promoted the migration of tumor cells. The number of tumor metastases in the lungs, induced by hypoxic TAM, was significantly higher than that of normoxic TAM. Hypoxia can significantly stimulate the expression of CCL22. CCL22 can significantly promote the migration of MDA-MB-231 and BT-549 cells. The expressions of CCR3, CCR4, and CCR5 in tumor tissues were significantly increased compared with normal tissues, in which CCR4 showed the most significant increase. Conclusion: TAM cultured under hypoxia significantly enhanced the migration ability of TNBC cells and promoted the metastasis of cancer cells to the lungs in vivo. The hypoxic condition induced TAM to secrete CCL22; the expression of CCL22 receptor, CCR4, in breast cancer tissues was significantly higher than that in normal tissues.

“Double-punch” strategy against triple-negative breast cancer via a synergistic therapy of magneto-mechanical force enhancing NIR-II hypothermal ablation

Triple-negative breast cancer (TNBC) is a form of breast cancer that is more aggressive and harder to treat than others, with a higher probability of relapse. Its nefarious capabilities for migrating and invading other parts of the body together with the current lack of clinically established effective therapies account for a low survival rate. In this work, we demonstrate the in-tandem use of two complementary therapeutic routes to effectively combat TNBC. A versatile magnetic-photothermal converter (MPC) consisting of zinc-doped ferrite nanoparticles and polyethene glycol, is shown to display excellent therapeutic efficiency, being capable to fight TNBC via two distinct routes: magneto-mechanical force (MMF) and near-infrared-II (NIR-II) hypothermal ablation. The combined use of these two complementary and synergistic therapies, which are less aggressive to the human body compared to conventional chemotherapeutic approaches, results in the splendid suppression of TNBC migration and invasion. Remotely controlling the MPCs by an external magnetic field, results in cellular MMF effects that cause direct mechanical destruction to the cancer cell membrane, leading to its necrosis. Furthermore, the MMF disrupts intracellular lysosomes, thereby triggering the release of large amounts of protein hydrolases, which induce intracellular oxidative stress, and accelerate the induction of apoptosis. Complementing the therapeutic approach based on MMF, the excellent photothermal performance of the MPC in the NIR-II region (1064 nm) is exploited to enable effective hypothermal ablation of the tumours, which can be achieved in deep tissue layers. The proposed multifunctional nanocomposites, together with the demonstrated “double-punch” therapeutic approach, hold significant potential to pave the way for future cutting-edge weapons against the dreadful TNBC.

XS-2, a novel potent dual PI3K/mTOR inhibitor, exhibits high in vitro and in vivo anti-breast cancer activity and low toxicity with the potential to inhibit the invasion and migration of triple-negative breast cancer

Breast cancer has become the most commonly diagnosed cancer, surpassing lung cancer, with 2.26 million new breast cancers worldwide in 2020. Hence, there is an urgent need to develop effective molecularly targeted therapeutic drugs to treat breast cancer. In this paper, we designed, synthesized and screened a novel thiophene-triazine derivative, XS-2, as a potent dual PI3K/mTOR inhibitor for the treatment of breast cancer. Also, XS-2 was found to be potentially effective against triple-negative breast cancer (TNBC) in vitro during the investigation. We evaluated the in vitro inhibitory effect of XS-2 on 10 cancer cell lines by MTT and 6 kinases to investigated its in vivo antitumor activity in MCF-7 xenograft tumor-bearing BALB/c nude mice. In addition, the in vitro/in vivo toxicity to mice was also assessed by hemolytic toxicity, H&E staining and blood biochemical analysis. In order to investigate the antitumor mechanism of XS-2, a series of experiments were carried out in vitro/in vivo animal model and molecular biological levels such as the cell cycle and the apoptosis assay, real-time PCR, western blot, docking and molecular simulations analysis, etc. What’s more, wound healing assay, Transwell and Western Blot were applied to explore the ability of XS-2 to inhibit the cell invasion and migration. The results showed that XS-2 exhibited strong antitumor activity both in vitro and in vivo. The inhibitory activities of XS-2 on ten cancer cell lines were ranging from 1.07 ± 0.11 to 0.002 ± 0.001 μM, which were 1565 times better than that of the lead compound GDC-0941, inhibitory activities against PI3Kα and mTOR kinases were 291.0 and 60.8 nM, respectively. Notably, XS-2 not only showed significant in vivo antitumor activity and low toxicity, with the tumor inhibition rate of 57.0 %, but also exhibited strong inhibitory in the expression of related proteins of PI3K pathway in tumor tissues. In addition, XS-2 significantly inhibited breast cancer MCF-7 and MDA-MB-231 cells in a concentration- and time-dependent manner, and inhibited the migration and invasion ability of MDA-MB-231 and MCF-7 cells. More than that, XS-2 could inhibit the increase of the expression levels of N-cadherin and vimentin upregulated by EGF and reversed the E-cadherin expression down regulated by EGF, resulting in inhibiting EMT in MCF-7 and MDA-MB-231 cells. The results showed that XS-2 was expected to be successfully developed as a high-efficiency and low-toxicity breast cancer therapeutic drug with the potential to inhibit the invasion and migration of TNBC. This provides a new research idea for the treatment of TNBC, which is of great significance.