Inhibits Breast Cancer Cell Migration Research Articles

Regulatory role of lncRNA MALAT1 and miR-150-3p interaction in breast cancer progression and therapeutic implications

Long non-coding RNAs (lncRNAs) play crucial roles in cancer progression and metastasis. However, the precise regulatory mechanisms of MALAT1, an lncRNA, in breast cancer remain elusive. Consequently, this study explored the specific role of MALAT1 as a tumor promoter in breast cancer. Initially, MALAT1-small interfering RNA (siRNA) transfection resulted in the inhibition of breast cancer cell migration, colony formation, and invasion. Real-time polymerase chain reaction analysis also revealed increased E-cadherin expression and decreased vimentin and vascular endothelial growth factor (VEGF) mRNA levels subsequent to the transfection. Bioinformatic analysis further uncovered a specific interaction between MALAT1 and miR-150-3p, indicating elevated MALAT1 expression in breast cancer with a negative correlation to miR-150-3p expression. Furthermore, the overexpression of MALAT1 markedly suppressed miR-150-3p expression, indicating a reciprocal inhibition between the two. Luciferase reporter assays confirmed the specific association between miR-150-3p and MALAT1 at the sequence level. Furthermore, transfection with miR-150-3p mimic inhibited breast cancer cell migration, colony formation, and invasion, along with increased E-cadherin expression and decreased Vimentin and VEGF mRNA levels. Conversely, miR-150-3p inhibitor transfection led to opposing effects, reducing E-cadherin expression, and elevating vimentin and VEGF mRNA levels, while also inhibiting migration, colony formation, and invasion. Functionally, MALAT1 siRNA restrained breast cancer cell proliferation and migration while enhancing cellular apoptosis on administration to breast cancer cells, primarily mediated through miR-150-3p. Our findings delineate MALAT1 as a tumor growth-promoting gene antagonized by miR-150-3p. Building on these insights, this study proposes a potential therapeutic avenue for breast cancer treatment.

Abstract LB034: First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy

Abstract We report the identification and complete characterization of first-in-class molecules targeting the MIEN1 signaling pathway in cancer. Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK-SCR and RP-7-SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo. Citation Format: Amit Kumar Tripathi, Jamboor K. Vishwanatha. First-in-class compounds targeting the MIEN1 signaling pathway: A breakthrough approach for breast and prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB034.

Short peptides based on the conserved regions of MIEN1 protein exhibit anticancer activity by targeting the MIEN1 signaling pathway

Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-κB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.

Curcumin for Treating Breast Cancer: A Review of Molecular Mechanisms, Combinations with Anticancer Drugs, and Nanosystems.

Breast cancer (BC) has become the fifth most prevalent cause of cancer-related morbidity, attracting significant attention from researchers due to its heightened malignancy and drug resistance. Conventional chemotherapy approaches have proven inadequate in addressing all BC subtypes, highlighting the urgent need for novel therapeutic approaches or drugs. Curcumin (CUR), a phytochemical derived from Curcuma longa (turmeric), has shown substantial potential in inhibiting BC cell migration, metastasis, and proliferation. However, the use of CUR in this context comes with challenges due to its dynamic and easily degradable nature, poor aqueous solubility, low bioavailability, rapid metabolism, and swift systemic elimination, collectively limiting its clinical applications. As such, we provide an overview of the properties, synthesis, and characterization of the hybridization of CUR and its analogue with chemo-drug building blocks. We reviewed research from the last five years on CUR's biogenesis with respect to the regulation of BC, revealing that CUR participates in arresting BC cells in the cell cycle and significantly induces apoptosis in BC cells. Information on the chemotherapeutic and antitumor mechanisms of CUR in BC, including regulation of the cell cycle, increased cell apoptosis, and inhibition of multidrug resistance (MDR), was compiled. Additionally, we provide an overview of CUR loaded into nanomaterials that are cotreated with other chemotherapeutic drugs, such as paclitaxel, thymoquinone, and tamoxifen. In this review, we discuss different types of nanoparticles that can be used for CUR delivery, such as polymeric nanoparticles, carbon nanotubes, and liposomes. By comparing the size, entrapment efficiency, drug-loading capacity, release time, biocompatibility, pharmaceutical scale, and reproducibility of various nanomaterials, we aimed to determine which formulations are better suited for loading CUR or its analogue. Ultimately, this review is expected to offer inspiring ideas, promising strategies, and potential pathways for developing advanced anti-BC strategy nanosystems in clinical practice.

Pigmented Microbial Extract (PMB) from Exiguobacterium Species MB2 Strain (PMB1) and Bacillus subtilis Strain MB1 (PMB2) Inhibited Breast Cancer Cells Growth In Vivo and In Vitro.

Breast cancer (BC) continues to be one of the major causes of cancer deaths in women. Progress has been made in targeting hormone and growth factor receptor-positive BCs with clinical efficacy and success. However, little progress has been made to develop a clinically viable treatment for the triple-negative BC cases (TNBCs). The current study aims to identify potent agents that can target TNBCs. Extracts from microbial sources have been reported to contain pharmacological agents that can selectively inhibit cancer cell growth. We have screened and identified pigmented microbial extracts (PMBs) that can inhibit BC cell proliferation by targeting legumain (LGMN). LGMN is an oncogenic protein expressed not only in malignant cells but also in tumor microenvironment cells, including tumor-associated macrophages. An LGMN inhibition assay was performed, and microbial extracts were evaluated for in vitro anticancer activity in BC cell lines, angiogenesis assay with chick chorioallantoic membrane (CAM), and tumor xenograft models in Swiss albino mice. We have identified that PMB from the Exiguobacterium (PMB1), inhibits BC growth more potently than PMB2, from the Bacillus subtilis strain. The analysis of PMB1 by GC-MS showed the presence of a variety of fatty acids and fatty-acid derivatives, small molecule phenolics, and aldehydes. PMB1 inhibited the activity of oncogenic legumain in BC cells and induced cell cycle arrest and apoptosis. PMB1 reduced the angiogenesis and inhibited BC cell migration. In mice, intraperitoneal administration of PMB1 retarded the growth of xenografted Ehrlich ascites mammary tumors and mitigated the proliferation of tumor cells in the peritoneal cavity in vivo. In summary, our findings demonstrate the high antitumor potential of PMB1.

Sulfated polysaccharides of Laetiporus sulphureus fruiting bodies exhibit anti-breast cancer activity through cell cycle arrest, apoptosis induction, and inhibiting cell migration

Ethnopharmacological relevanceLaetiporus sulphureus has long been used as an edible and medicinal mushroom in Asia, America, and Europe. Its fruiting bodies are widely used in folk medicine for treating cancer, gastric diseases, cough, and rheumatism. Polysaccharides are an important bioactive component of mushrooms. In nature, sulfated polysaccharides have never been reported in mushrooms. Furthermore, there is no information on differences in physicochemical properties and anti-breast cancer activities between polysaccharides (PS) and sulfated polysaccharides (SPS) of L. sulphureus. Aim of the studyThis study aimed to investigate the physicochemical properties of PS and SPS isolated from fruiting bodies of L. sulphureus and examine their anti-proliferative effects and mechanism(s) of action on MDA-MB-231 breast cancer cells. MethodsPolysaccharides (PS) were isolated using hot water and ethanol precipitation methods. Sulfated polysaccharides (SPS) were isolated by the papain-assisted hydrolysis method. Physicochemical properties comprising sugar, protein, uronic acid, and sulfate contents, and molecular weight, monosaccharide composition, and structural conformation were analyzed on PS and SPS. In the anti-cancer study, a triple-negative breast cancer cell line (MDA-MB-231) and a normal human mammary epithelial cell line (H184B5F5/M10) were used to evaluate the anti-proliferative activity of PS and SPS, and their mechanism(s) of action. ResultsThe results showed that SPS, which had higher sulfate and protein contents and diversified monosaccharide composition, exhibited more potent anti-proliferative activity against MDA-MB-231 cells than PS. Furthermore, it had a selective cytotoxic effect on breast cancer cells but not the normal cells. SPS induced cell cycle arrest at G0/G1 phase via down-regulating CDK4 and cyclin D1 and up-regulating p21 protein expression. Breast cancer cell apoptosis was not observed until 72 h after SPS treatment. In addition, SPS also markedly inhibited breast cancer cell migration. ConclusionThis study demonstrates that SPS exhibited selective cytotoxicity and was more potent than PS in inhibiting MDA-MB-231 cell proliferation. The contents of sulfate and protein, and monosaccharide composition could be the main factors affecting the anti-breast cancer activity of L. sulphureus SPS.

Macrophage-secreted exosomes inhibit breast cancer cell migration via the miR- 101-3p/DLG5 axis

Objective: To investigate the role of macrophages in regulating breast cancer cell migration and its related mechanisms. Methods: Human leukemia monocytic cell line THP-1-secreted exosomes were isolated using multi-step ultracentrifugation and verified using nanoparticle tracking analysis. Differentially expressed miRNAs were identified using RNA sequencing. Overexpression of inhibitors of hsa-miR-101-3p in breast cancer MDA-MB-231 cells was performed by infecting their lentiviral constructs. The luciferase reporter assay was used to evaluate the interaction of DLG5 and miR-101. DGL5 expression was detected using qRT-PCR and Western blot analyses. Results: The migration of breast cancer cells was significantly inhibited after addition of exosomes. RNA sequencing results showed that miR-101-3p expression was significantly upregulated. Targetscan analysis predicted that miR-101-3p could target DLG5, and this prediction was verified using the luciferase assay. The addition of the miR-101-3p precursor significantly increased the expression of miR-101-3p, and the mRNA and protein levels of DLG5 were suppressed. In contrast, inhibiting the expression of miR-101-3p increased the mRNA and protein levels of DLG5. Furthermore, the scratch assay showed that inhibiting miR-101-3p could promote the migration of MDA-MB-231 cells. Conclusions: Macrophage exosomes can inhibit the migration of breast cancer cells, and increasing the expression of miR-101-3p to inhibit DLG5 expression may play an important role in this process, which needs further investigation.

Different Modes of Mechanism of Gamma-Mangostin and Alpha-Mangostin to Inhibit Cell Migration of Triple-Negative Breast Cancer Cells Concerning CXCR4 Downregulation and ROS Generation.

Two mangostin compounds, gamma-mangostin and alpha-mangostin, show anticancer properties through the inhibition of cell proliferation and cell migration. Metastatic triple-negative breast cancer (TNBC) cells, including MDA-MB-231, highly express C-X-C chemokine receptor type 4 (CXCR4) to maintain reactive oxygen species (ROS) and cell migration. This study was performed to analyze and compare different modes of action of γ-mangostin and α-mangostin as antimigratory effects targeted on CXCR4 in MDA-MB-231 as a model of TNBC cell. This study investigated the effect of γ-mangostin and α-mangostin using a series of assays, including Cell Counting Kit-8 (CCK-8) assay for cytotoxicity, wound healing assay for migration study, quantitative real-time polymerase chain reaction (qRT-PCR) for gene expression analysis, and flow cytometry for ROS measurement, along with in silico study to observe the binding between the compound and CXCR4. The findings revealed half maximal inhibitory concentration (IC50) values of 25 and 20 μM for γ-mangostin and α-mangostin in MDA-MB 231 cells, respectively. Moreover, a concentration of 10 μM was used for the migration assay. Both γ-mangostin and α-mangostin significantly suppressed cell migration within 24 hours. The present gene expression studies revealed the downregulation of key migration-associated genes, namely Farp, CXCR4, and LPHN2, upon γ-mangostin treatment but not α-mangostin. Additionally, both γ-mangostin and α-mangostin increased cellular ROS generation, highlighting the same effect of γ-mangostin and α-mangostin ROS elevation to inhibit cancer cell migration. Molecular docking simulations further suggested a potential interaction between γ-mangostin and α-mangostin with CXCR4 in high affinity. These findings suggest that both γ-mangostin and α-mangostin inhibit breast cancer cell migration and induce cellular ROS levels in MDA-MB-231 cells; notably, γ-mangostin suppresses CXCR4 mRNA expression that might correlate to its activity to inhibit MDA-MB-231 cell migration.

Oridonin impedes breast cancer growth by blocking cells in S phase and inhibiting the PI3K/AKT/mTOR signaling pathway

Breast cancer is one of the most common cancers. Oridonin, a traditional Chinese medicine, is believed to inhibit tumor growth, but its particular effects on breast cancer remain unknown. In this study, we examined oridonin's effects on 4T1, MCF-7, and MDAMB-231 cellular activity using CCK8. Scratch assays were used to detect oridonin's effects on cellular migration. Oridonin's effects on the breast cancer cell cycle were studied using flow cytometry, and expression of cell cycle related proteins p53, CDK2, and p21 was detected using Western blot assays. Metabolomics assays were used to detect changes in small molecule metabolites and metabolic pathways in breast cancer cells after treatment with oridonin. Oridonin's effects on breast cancer growth were also studied using xenograft mice. Metabolomics assays were used to detect changes in metabolites and metabolic pathways in xenograft mouse plasma in a control group, model group, and drug administration group. Experimental results showed that oridonin could significantly inhibit breast cancer growth both in vivo and in vitro. Scratch experiments showed that oridonin could inhibit breast cancer cell migration. Oridonin was also able to arrest cells in S phase by affecting several cell cycle-related proteins, including p53, CDK2, and p21. Metabolomic analysis of 4T1 cells identified a total of 33 differential metabolites, including multiple amino acids (such as l-Glutamic acid, l-Asparagine, l-Histidine, l-Valine, and l-Isoleucine). KEGG pathway enrichment analysis showed significant changes in aminoacyl-tRNA biosynthesis, and in multiple amino acid metabolic pathways. Plasma metabolomic analyses of xenograft mice revealed 28 differentially-expressed metabolites between the different animal model groups, including multiple amino acids. KEGG pathway analysis showed significant alterations in multiple amino acid metabolic pathways in oridonin-treated mice. Additionally, changes in the expression of PI3K, AKT and mTOR proteins, as well as in branched amino acids, suggest that oridonin affects the PI3K/AKT/mTOR signaling pathway by inhibiting the biosynthesis of valine, leucine and isoleucine. Taken together, our results suggest that oridonin has strong anti-tumor activity in vitro and in vivo, and has potential as an adjuvant to breast cancer treatment regimens.

Neoprzewaquinone A Inhibits Breast Cancer Cell Migration and Promotes Smooth Muscle Relaxation by Targeting PIM1 to Block ROCK2/STAT3 Pathway

Salvia miltiorrhiza Bunge (Danshen) has been widely used to treat cancer and cardiovascular diseases in Chinese traditional medicine. Here, we found that Neoprzewaquinone A (NEO), an active component of S. miltiorrhiza, selectively inhibits PIM1. We showed that NEO potently inhibits PIM1 kinase at nanomolar concentrations and significantly suppresses the growth, migration, and Epithelial-Mesenchymal Transition (EMT) in the triple-negative breast cancer cell line, MDA-MB-231 in vitro. Molecular docking simulations revealed that NEO enters the PIM1 pocket, thereby triggering multiple interaction effects. Western blot analysis revealed that both NEO and SGI-1776 (a specific PIM1 inhibitor), inhibited ROCK2/STAT3 signaling in MDA-MB-231 cells, indicating that PIM1 kinase modulates cell migration and EMT via ROCK2 signaling. Recent studies indicated that ROCK2 plays a key role in smooth muscle contraction, and that ROCK2 inhibitors effectively control the symptoms of high intraocular pressure (IOP) in glaucoma patients. Here, we showed that NEO and SGI-1776 significantly reduce IOP in normal rabbits and relax pre-restrained thoracic aortic rings in rats. Taken together, our findings indicated that NEO inhibits TNBC cell migration and relaxes smooth muscles mainly by targeting PIM1 and inhibiting ROCK2/STAT3 signaling, and that PIM1 may be an effective target for IOP and other circulatory diseases.

Synthesis and bioassay of 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones as anti-cancer agents.

Four novel 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones derivatives (C1 to C4) have been designed, synthesized, and evaluated for their anticancer activity. The structure of compounds was characterized by IR,1H NMR, 13C NMR and high-resolution mass (HRMS). The crystal structures of C1, C2 and C4 were previously determined by single-crystal X-ray analysis.The results from docking experiments with EGFR suggested the binding of the compounds at the active site of EGFR. The new compounds exhibited different levels of cytotoxicity against HCC1937 and MCF7 breast cancer cells. Results of the MTT assay identified C3 as the most cytotoxic of the series against both MCF7 and HCC1937 breast cancer cell lines with IC50 values of 36.4 and 48.2 µM, respectively. In addition to its ability to inhibit cell growth and colony formation ability, C3 also inhibited breast cancer cell migration. Western blotting results showed that C3 treatment inhibited EGFR signaling and induced cell cycle arrest and apoptosis as indicated by the low level of p-EGFR and p-AKT and the increasing levels of p53, p21 and cleaved PARP. Our work represents a promising starting point for the development of a new series of compounds targeting cancer cells.

Sini San inhibits breast cancer cell migration and angiogenesis via the HIF 1 /VEGF pathway

Purpose: To investigate the effects of Sini San (SNS) on breast cancer (BC), and the mechanism of action.Methods: MDA-MB-231 and SK-BR-3 cells were used as breast cancer cell models. Cell viability, migration, and invasion were determined by CCK-8, Transwell and wound healing assays, respectively. SNS mechanism of action and its anti-cancer effect were investigated by network pharmacological analysis, and further verified by Immunoblot.Results: Sini San inhibited the proliferation of the breast cancer (BC) cells., and also suppressed the migration as well as the invasion of BC cells, and also restrained the angiogenesis of BC cells. In performing the network pharmacological analysis of Sini Powder in the treatment of BC, 337 drugdisease targets were obtained. PPI network was established through String, and GO and KEGG enrichment analysis was performed on the target sites. KEGG analysis showed that genes were enriched in HIF-1 and VEGF pathways.Conclusion: Sini San suppressed cell migration as well as angiogenesis via the HIF-1 /VEGF pathway

CBX3 promotes breast cancer progression and high level of CBX3 predicts poor prognosis in patients.

Breast cancer is one of the leading cancer deaths around the world. Targeted drugs have greatly increased the survival rate of breast cancer patients in recent years. But in some patients, the current regimen is still ineffective. Therefore, more therapeutic targets for treating breast cancer are demanding. The core heterochromatin-related genes of breast cancer were identified by utilizing prognostic survival analysis and multivariate Cox hazard proportional regression analysis. Both breast cancer and adjacent normal tissue were collected and analyzed with western blot and immunohistochemistry. Colony formation assay, CCK-8 assay, and EdU assay were used to measure the effect of CBX3 on breast cancer cell growth, wound-healing assay and Transwell assay were used to analyze the effect of CBX3 on breast cancer cell migration and invasion. Flow cytometry assay and western blot were used to study the molecular mechanism of CBX3 in breast cancer. High expression of heterochromatin-related proteins CBX3, H2AFY, and SULF1 showed a poor prognosis in patients in both TCGA dataset and GEO datasets. Western blot demonstrated that the expression level of CBX3 was significantly higher in breast cancer than that in adjacent normal tissues. Colony formation assay, CCK-8 assay, and EdU assay showed that the knockdown of CBX3 could significantly inhibit breast cancer cell growth, and the overexpression of CBX3 could promote the growth of breast cancer cells. Transwell assay and wound healing assay showed that knockdown of CBX3 inhibited breast cancer cell migration and invasion, and the overexpression of CBX3 promoted breast cancer cell migration and invasion. Western blot showed that CBX3 might promote breast cancer cell proliferation, invasion, and migration in breast cancer by modulating the ERK1/2 signaling pathway and epithelial-mesenchymal transition (EMT)-related genes. CBX3 was a biomarker of poor prognosis in breast cancer patients. CBX3 promoted the proliferation of breast cancer cells through the ERK signaling pathway, and migration and invasion of breast cancer cells through EMT-related genes. The CBX3/p-ERK1/2 signaling axis might provide a new therapeutic method against breast cancer.

Nobiletin inhibits breast cancer cell migration and invasion by suppressing the IL-6-induced ERK-STAT and JNK-c-JUN pathways

BackgroundBreast cancer is one of the most common cancers in women, affecting more than 2 million women worldwide annually. However, effective treatments for breast cancer are limited. Nobiletin is a flavonoid present in the dried mature pericarp of mandarin orange (Citrus reticulata Blanco), which is used to prepare Citri Renetulatae Pericarpium and can inhibit tumour growth and progression according to modern pharmacological studies. However, whether nobiletin exhibits an antimetastatic role in breast cancer and its potential mechanism need to be further investigated. PurposeThis study aims to evaluate the inhibitory effect of nobiletin on breast cancer and to elucidate potential mechanisms against invasion and migration. MethodsCell viability was determined by cell counting kit-8 and colony formation assays. Wound healing and Boyden chamber assays detected cancer cell migration and invasion capabilities. Immunoblotting and qPCR were applied to determine the protein and mRNA expression levels of extracellular signal-regulated kinases (ERK) and the c-Jun N-terminal kinase (JNK) signalling pathways. Molecular docking was used to assess the degree of nobiletin binding to phosphatidylinositol 3-kinase (PI3K). Xenografts and liver metastases were constructed in BALB/c nude mice to evaluate the anticancer effect of nobiletin in vivo. H&E staining and immunohistochemistry were used to detect proliferation and the expression of related proteins. ResultsNobiletin induced cell death in a concentration- and time-dependent manner and possessed anti-invasion and anti-migration effects on MCF-7 and T47D cells by suppressing the interleukin-6-induced ERK and JNK signalling pathways. In addition, nobiletin docked with the binding site of PI3K, and the binding score was -8.0 kcal/mol. Furthermore, the inhibition of breast cancer growth and metastasis by nobiletin was demonstrated by constructing xenografts and liver metastases in vivo. ConclusionNobiletin inhibited liver metastasis of breast cancer by downregulating the ERK-STAT and JNK-c-JUN pathways, and its safety and efficacy were verified, indicating the potential of nobiletin as an anticancer agent.

Kaurenoic acid nanocarriers regulates cytokine production and inhibit breast cancer cell migration.

Breast cancer is the type of cancer with the highest incidence in women around the world. Noteworthy, the triple-negative subtype affects 20% of the patients while presenting the highest death rate among subtypes. This is due to its aggressive phenotype and the capability of invading other tissues. In general, tumor-associated macrophages (TAM) and other immune cells, are responsible for maintaining a favorable tumor microenvironment for inflammation and metastasis by secreting several mediators such as pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, chemokines like CCL2, and other proteins, as metalloproteinases of matrix (MMP). On the other hand, immunomodulatory agents can interfere in the immune response of TAM and change the disease prognosis. In this work, we prepared nanostructured lipid carriers containing kaurenoic acid (NLC-KA) to evaluate the effect on cytokine production in vitro of bone marrow-derived macrophages (BMDM) and the migratory process of 4T1 breast cancer cells. NLC-KA prepared from a blend of natural lipids was shown to have approximately 90nm in diameter with low polydispersity index. To test the effect on cytokine production in vitro in NLC-KA treated BMDM, ELISA assay was performed and pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified. The formulation reduced the secretion of IL-1β and TNF-α cytokines while presenting no hemolytic activity. Noteworthy, an anti-migratory effect in 4T1 breast cancer cells treated with NLC-KA was observed in scratch assays. Further, MMP9 and CCL2 gene expressions in both BMDM and 4T1 treated cells confirmed that the mechanism of inhibition of migration is related to the blockade of this pathway by KA. Finally, cell invasion assays confirmed that NLC-KA treatment resulted in less invasiveness of 4T1 cells than control, and it is independent of CCL2 stimulus or BMDM direct stimulus. Ultimately, NLC-KA was able to regulate the cytokine production in vitro and reduce the migration of 4T1 breast cancer cells by decreasing MMP9 gene expression.

Histone demethylase KDM2A suppresses EGF-TSPAN8 pathway to inhibit breast cancer cell migration and invasion in vitro

Metastasis is a major cause of breast cancer mortality and the current study found histone demethylase, KDM2A, expression to be negatively correlated with breast cancer metastasis. KDM2A knockdown greatly promoted migration and invasion of breast cancer cells. The histone demethylase activity of KDM2A downregulated EGF transcription and suppressed the EGF-TSPAN8 pathway. Inhibition of breast cancer cell migration was also dependent on the histone demethylase activity of KDM2A. A novel mechanism of KDM2A-suppression of the EGF-TSPAN8 pathway which inhibited breast cancer cell migration and invasion is reported.

Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

SummaryCleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation. Recent work has surprisingly identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers, triggering further studies on understanding CPSF73 regulation and functions in cells. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression in cells. UBE3D dysfunction or chemical inactivation of CPSF73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple-negative breast cancer cells. In addition, genetic overexpression of CPSF73 promoted breast cancer stemness and knocking down CPSF73 inhibited stem cell renewal properties. Together, our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy.

CLDN6 inhibits breast cancer cell malignant behavior by suppressing ERK signaling

Claudin 6 (CLDN6) is an important component of tight junctions. Through the PDZ binding motif, CLDN6 binds to a variety of signaling proteins that contain the PDZ domain to regulate different signaling pathways, and plays an important role in the occurrence and development of tumors. Our previous work showed that CLDN6 was expressed at low levels in breast cancer cells, and overexpression of CLDN6 inhibited breast cancer cell proliferation, migration and invasion. However, the mechanism of how CLDN6 works remains unclear. In this study, we aimed to explore the mechanism by which CLDN6 inhibits breast cancer cell malignant behavior. As a result, overexpression of CLDN6 inhibited the proliferation of breast cancer cells along with the downregulation of cyclin D1, which plays an important role in regulating cell proliferation. After overexpression of Sp1 in CLDN6-overexpressing cells, the expression of cyclin D1 was upregulated. On the other hand, CLDN6 inhibited breast cancer cell migration and invasion along with the downregulation of IL-8, CXCR2 and FAK. When treated with IL-8, the migration and invasion ability were promoted along with the upregulation of CXCR2 and p-FAK, and the cytoskeleton was rearranged in CLDN6-overexpressing cells. Furthermore, when treated with the ERK signaling activator PMA, the proliferation, migration and invasion abilities were promoted along with the upregulation of Sp1, cyclin D1 and IL-8 in CLDN6-overexpressin cells. In conclusion, CLDN6 suppressed ERK/Sp1/cyclin D1 and ERK/IL-8 signaling to inhibit proliferation, migration and invasion in breast cancer cells. The mechanism may provide experimental evidence for the treatment of breast cancer targeting CLDN6.

Abstract 5722: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration and invasion

Abstract Cleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation, and abnormal poly(A) site choices have been associated with human diseases, including cancer. Recent work has identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers, and higher level of CPSF73 correlates to poor survival in breast cancer patients. However, our understanding of the regulation of CPSF73 is still at an early stage. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D in both non-cancer cells and cancer cells leads to CPSF73 downregulation, a pre-mRNA cleavage defect and dysregulated gene expression. UBE3D dysfunction or chemical inactivation of CPFS73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple negative breast cancer cells. In addition, genetic overexpression of CPSF73 promoted breast cancer stemness and knocking down of CPSF73 inhibited stem cell renewal property. Together, our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy. This work was supported by the NSF grant NS0224 to C. Moore and the NIH grant R01 GM101010-01A1 to C. Moore. Citation Format: Huiyun Liu, Daniel Heller-Trulli, Claire Moore. Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5722.

Carnosol Induces p38-Mediated ER Stress Response and Autophagy in Human Breast Cancer Cells.

We recently reported that carnosol induces ROS-dependent autophagy and apoptosis in breast cancer cells. We also reported that carnosol inhibits breast cancer cell migration, invasion, and in ovo tumor growth, as well as targets STAT3, PCAF, and p300 to proteasome degradation. Here, we investigated the molecular mechanisms underlying its anti-malignant activity in breast cancer. We report that carnosol induces a ROS-dependent type I and type II programmed cell death (PCD-I or PCD-II, respectively), which occurred independently of each other. Indeed, chemical inhibition of autophagy had no effect on the induction of apoptosis, evident by the absence of cleaved PARP. Electron microscopy revealed that carnosol-treated cells exhibited enlarged endoplasmic reticulum, characteristic of ER stress. Markers of the three unfolded protein response pathways (PERK, IRE-1 α, and ATF6), namely ATF4, CHOP, phospho-IRE-1α, XBP1S, and cleaved ATF6 were upregulated in a ROS-dependent manner. In addition, carnosol induced a ROS-dependent activation of p38MAPK, increased the overall level of protein polyubiquitination, and targeted mTOR protein to proteasome degradation. Interestingly, inhibition of p38MAPK, by SB202190 and 203580, reduced cell death, selectively blocked the induction of IRE-1α and ATF6 UPR sensors and inhibited autophagy. In addition, inhibition of p38 reduced the carnosol-induced polyubiquitination and rescued mTOR, PCAF, and STAT3 from proteasomal degradation. Importantly, activation of PERK sensors and induction of apoptosis occurred independently of p38 activation. Taken together, our results suggest that ROS-dependent induced-ER stress contributes to carnosol-induced apoptotic and autophagic cell death in breast cancer cells, and further confirm that carnosol is a promising agent for breast cancer therapy.