Metastasis Of Breast Cancer Cells Research Articles

Retraction Note: HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs.

Retraction Note: HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs.

The role of peptides in reversing chemoresistance of breast cancer: current facts and future prospects.

Breast cancer is the first malignant tumor in women, and its incidence is also increasing year by year. Chemotherapy is one of the standard therapies for breast cancer, but the resistance of breast cancer cells to chemotherapy drugs is a huge challenge for the effective treatment of breast cancer. At present, in the study of reversing the drug resistance of solid tumors such as breast cancer, peptides have the advantages of high selectivity, high tissue penetration, and good biocompatibility. Some of the peptides that have been studied can overcome the resistance of tumor cells to chemotherapeutic drugs in the experiment, and effectively control the growth and metastasis of breast cancer cells. Here, we describe the mechanism of different peptides in reversing breast cancer resistance, including promoting cancer cell apoptosis; promoting non-apoptotic regulatory cell death of cancer cells; inhibiting the DNA repair mechanism of cancer cells; improving the tumor microenvironment; inhibiting drug efflux mechanism; and enhancing drug uptake. This review focuses on the different mechanisms of peptides in reversing breast cancer drug resistance, and these peptides are also expected to create clinical breakthroughs in promoting the therapeutic effect of chemotherapy drugs in breast cancer patients and improving the survival rate of patients.

Collagen-induced DDR1 upregulates CXCL5 to promote neutrophil extracellular traps formation and Treg infiltration in breast cancer

Neutrophil extracellular traps (NETs) have been implicated in many cancers, but the regulatory mechanisms in the context of breast cancer have not been thoroughly discussed. This study proposed a mechanism based on collagen-activated DDR1/CXCL5 for NET formation in breast cancer. Through TCGA and GEO-based bioinformatics analysis, we examined the DDR1 expression and the correlation of CXCL5 with immune cell infiltration in breast cancer. It was found that high DDR1 expression was correlated with poor prognosis of patients with breast cancer, and CXCL5 was positively correlated with neutrophil and Treg infiltration. Expression of DDR1 and CXCL5 was determined in collagen-treated breast cancer cells, the malignant phenotypes of which were evaluated by ectopic expression and knockdown methods. Collagen-activated DDR1 upregulated CXCL5 expression, resulting in augmented malignant phenotypes of breast cancer cells in vitro. The formation of NETs caused promotion in the differentiation and immune infiltration of Tregs in breast cancer. A in situ breast cancer mouse model was constructed, where NET formation and lung metastasis of breast cancer cells were observed. The differentiation of CD4+ T cells isolated from the mouse model was induced into Tregs, followed by Treg infiltration assessment. It was further confirmed in vivo that DDR1/CXCL5 induced the formation of NETs to promote immune infiltration of Tregs, driving tumor growth and metastasis. Accordingly, our results provided new mechanistic insights for an understanding of the role of collagen-mediated DDR1/CXCL5 in formation of NETs and Treg infiltration, revealing potential targets for therapeutic intervention of breast cancer.

The relationship between the Hippo signaling pathway and bone metastasis of breast cancer.

Bone is the most common site of metastasis from breast cancer, which is the most prevalent cancer affecting women globally. Bone metastasis from breast cancer severely affects the quality of life of patients and increases mortality. The molecular mechanisms of metastasis, colonization, and proliferation of breast cancer cells in bone are complex and involve the interaction between breast cancer cells and the bone microenvironment. However, the precise mechanism is not clear at present. In recent years, the Hippo signaling pathway has attracted much attention due to its important role in regulating the expression of major effector molecules during tumor development. In particular, studies have found that the mutation and aberrant expression of the core components of the Hippo signaling pathway affect breast cancer cell migration and invasion, indicating that this pathway plays a role in bone metastasis, although the molecular mechanism of this pathway in breast cancer metastasis has not been fully elucidated. In this review, we discuss the function of the Hippo signaling pathway, introducing its role in breast cancer metastasis, especially bone metastasis of breast cancer, so as to lay a solid theoretical foundation for further research and for the development of effective targeted therapeutic agents.

Small peptide LINC00511-133aa encoded by LINC00511 regulates breast cancer cell invasion and stemness through the Wnt/β-catenin pathway

LINC00511 is an long non-coding RNA (lncRNA) of ncRNAs,This study aimed to investigate whether the lncRNA LINC00511 could encode a small peptide, LINC00511-133aa, and whether this peptide could promote breast cancer cell metastasis and stemness by activating the wnt/β-catenin pathway. The LINC00511-133aa coding sequence vector and control vector were transfected into MCF-7 and MDA-MB-231 breast cancer cells, with subsequent assessment of peptide expression using PCR, western blotting, and immunofluorescence assays. Cell proliferation, invasion, and apoptosis were evaluated using CCK8, apoptotic, wound healing, and transwell invasion assays, while the characteristic changes of tumor stem cells were detected through sphere-forming assay and western blot analyses of the stemness markers Oct4, Nanog, and SOX2. Results showed that LINC00511-133aa was indeed encoded by LINC00511 and promoted the invasiveness and stemness of breast cancer cells while limiting apoptosis by modulating the expression levels of wnt/β-catenin pathway-related proteins Bax, c-myc, and CyclinD1, as well as facilitating β-catenin protein entry into the nucleus. This study provides evidence for the potential involvement of lncRNA LINC00511 and its peptide product in breast cancer progression via the regulation of the wnt/β-catenin pathway.

CHST2-mediated sulfation of MECA79 antigens is critical for breast cancer cell migration and metastasis

Snail is a denoted transcriptional repressor that plays key roles in epithelial-mesenchymal transition (EMT) and metastasis. Lately, a plethora of genes can be induced by stable expression of Snail in multiple cell lines. However, the biological roles of these upregulated genes are largely elusive. Here, we report identification of a gene encoding the key GlcNAc sulfation enzyme CHST2 is induced by Snail in multiple breast cancer cells. Biologically, CHST2 depletion results in inhibition of breast cancer cell migration and metastasis, while overexpression of CHST2 promotes cell migration and lung metastasis in nude mice. In addition, the expression level of MECA79 antigen is elevated and blocking the cell surface MECA79 antigen with specific antibodies can override cell migration mediated by CHST2 upregulation. Moreover, the sulfation inhibitor sodium chlorate effectively inhibits the cell migration induced by CHST2. Collectively, these data provide novel insights into the biology of Snail/CHST2/MECA79 axis in breast cancer progression and metastasis as well as potential therapeutic strategy for the diagnosis and treatment of breast cancer metastasis.

Enhance Mitochondrial Damage by Nuclear Export Inhibition to Suppress Tumor Growth and Metastasis with Increased Antitumor Properties of Macrophages.

Mitochondria-targeting damage has become a popular therapeutic option for tumor metastasis; however, its efficacy is limited by the adaptive rescue capacity of nuclei. There is an urgent need for a dual mitochondrial and nuclear targeting strategy that can also increase the antitumor capacity of macrophages. In this study, XPO1 inhibitor KPT-330 nanoparticles were combined with mitochondria-targeting lonidamine (TPP-LND) nanoparticles. The combination of nanoparticles with a 1:4 ratio of KPT and TL demonstrated the best synergistic effect in restraining the proliferation and metastasis of 4T1 breast cancer cells. Investigating the mechanisms both in vitro and in vivo, it was found that KPT nanoparticles not only directly impede tumor growth and metastasis by controlling the expression of associated proteins but also indirectly facilitate mitochondrial damage. The two nanoparticles synergistically decreased the expression of cytoprotective factors, such as Mcl-1 and Survivin, causing mitochondrial dysfunction and thus inducing apoptosis. Additionally, it downregulated metastasis-related proteins like HIF-1α, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 2 (MMP-2) and reduced endothelial-to-mesenchymal transition. Significantly, their combination increased the ratio of M1 tumor-associated macrophages (TAMs)/M2 TAMs both in vitro and in vivo and increased the phagocytosis of tumor cells by macrophages, thus suppressing tumor growth and metastasis. In summary, this research revealed that nuclear export inhibition can synergistically enhance the prevention of mitochondrial damage to tumor cells, heightening the antitumor properties of TAMs, thereby providing a viable and safe therapeutic approach for the treatment of tumor metastasis.

Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas and complex fluids, which may lead to numerous plasma membrane damages RESULTS: : Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis. This article is protected by copyright. All rights reserved.

Targeting of Tetraspanin CD81 with Monoclonal Antibodies and Small Molecules to Combat Cancers and Viral Diseases

Tetraspanin CD81 plays major roles in cell-cell interactions and the regulation of cellular trafficking. This cholesterol-embarking transmembrane protein is a co-receptor for several viruses, including HCV, HIV-1 and Chikungunya virus, which exploits the large extracellular loop EC2 for cell entry. CD81 is also an anticancer target implicated in cancer cell proliferation and mobility, and in tumor metastasis. CD81 signaling contributes to the development of solid tumors (notably colorectal, liver and gastric cancers) and has been implicated in the aggressivity of B-cell lymphomas. A variety of protein partners can interact with CD81, either to regulate attachment and uptake of viruses (HCV E2, claudin-1, IFIM1) or to contribute to tumor growth and dissemination (CD19, CD44, EWI-2). CD81-protein interactions can be modulated with molecules targeting the extracellular domain of CD81, investigated as antiviral and/or anticancer agents. Several monoclonal antibodies anti-CD81 have been developed, notably mAb 5A6 active against invasion and metastasis of triple-negative breast cancer cells. CD81-EC2 can also be targeted with natural products (trachelogenin and harzianoic acids A-B) and synthetic compounds (such as benzothiazole-quinoline derivatives). They are weak CD81 binders but offer templates for the design of new compounds targeting the open EC2 loop. There is no anti-CD81 compound in clinical development at present, but this structurally well-characterized tetraspanin warrants more substantial considerations as a drug target.

Let-7b-5p inhibits breast cancer cell growth and metastasis via repression of hexokinase 2-mediated aerobic glycolysis

Hexokinase 2 (HK2), a critical rate-limiting enzyme in the glycolytic pathway catalyzing hexose phosphorylation, is overexpressed in multiple human cancers and associated with poor clinicopathological features. Drugs targeting aerobic glycolysis regulators, including HK2, are in development. However, the physiological significance of HK2 inhibitors and mechanisms of HK2 inhibition in cancer cells remain largely unclear. Herein, we show that microRNA-let-7b-5p (let-7b-5p) represses HK2 expression by targeting its 3′-untranslated region. By suppressing HK2-mediated aerobic glycolysis, let-7b-5p restrains breast tumor growth and metastasis both in vitro and in vivo. In patients with breast cancer, let-7b-5p expression is significantly downregulated and is negatively correlated with HK2 expression. Our findings indicate that the let-7b-5p/HK2 axis plays a key role in aerobic glycolysis as well as breast tumor proliferation and metastasis, and targeting this axis is a potential therapeutic strategy for breast cancer.

Pleckstrin-2-promoted PPM1B degradation plays an important role in transforming growth factor-β-induced breast cancer cell invasion and metastasis.

Transforming growth factor-β (TGF-β) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF-β induces pleckstrin-2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA-MB-231 and MCF-7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin-2 localizes to the cell membrane and cell protrusions following TGF-β treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF-β- and PLEK2-induced cell invasion. The carboxyl-terminal PH domain of PLEK2 is critical for TGF-β- and PLEK2-induced Akt activation and plays an important role in cell invasion. Pleckstrin-2 interacts with PPM1B and promotes its ubiquitin-dependent degradation. The PLEK2-PPM1B axis utilizes nuclear factor-κB signaling to promote cell migration and invasion. Our data implicate the TGF-β-STAT3/Smad3-PLEK2-PPM1B signaling cascade in TGF-β-induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.

ZNF32 prevents the activation of cancer-associated fibroblasts through negative regulation of TGFB1 transcription in breast cancer.

Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancer-related deaths in women worldwide. Cancer-associated fibroblasts (CAFs) are one of the fundamental cellular components of the tumor microenvironment and play a critical role in the initiation, progression, and therapy resistance of breast cancer. However, the detailed molecular mechanisms of CAFs activation from normal fibroblasts (NFs) are still not well understood. In the present study, we reported that ZNF32 expression in breast cancer cells was negatively correlated with CAF-related markers (FSP1, α-SMA, and FAP) in stromal fibroblasts, and loss of ZNF32 promoted the activation of CAFs, as evidenced by the enhanced proliferation and contractility of CAFs. ZNF32 deficiency-mediated fibroblast activation promoted the growth and metastasis of breast cancer cells in vitro and in vivo. Mechanistically, we demonstrated that ZNF32 inhibited TGFB1 transcription by directly binding to the -1968/-1962 region of the TGFB1 promoter, leading to the prevention of fibroblast activation. Altogether, our findings reveal an important mechanism by which ZNF32 suppression increases the transcription of the TGFB1 gene in breast cancer cells, and subsequently, elevated levels of secretory TGF-β stimulate NFstransformation into CAFs, which in turn facilitates the malignant progression of breast cancer. Our data implicated ZNF32 as a potential therapeutic strategy against breast cancer.

Abstract P6-12-03: Proinflammatory and estrogen signaling modulates the chemoresistance and metastasis of breast cancer cells through post-translational modifications of pioneering factor FOXA1

Abstract ER-positive breast cancers compose most breast cancers at the time of diagnosis and are primarily driven by mitogenic estrogen signaling. In ER-positive breast cancers, the pioneer transcription factor FOXA1 plays a critical role in the estrogen receptor (ER) function. It binds to condensed chromatin and promotes chromatin accessibility for subsequent ER binding upon estrogen stimulation. We have reported that TNFa-stimulated proinflammatory signaling relocates FOXA1 to a new set of latent enhancers, which initiates the binding of estrogen liganded ER and subsequent expression of a unique transcriptome with clinical significance. The redistribution of FOXA1 occurs within 40 mins of the TNFa treatment, which implies a rapid signaling cascade that arises from changes to either FOXA1’s post-translational modifications (PTMs) or its binding partners. To understand this genomic redistribution of FOXA1, we compared the post-translational modifications (PTMs) of FOXA1 from Vehicle, E2, TNFa, and E2+TNFa treated MCF-7 breast cancer cells. More than five acetylation and phosphorylation events have been identified around the DNA binding domain of FOXA1 by semi-quantitative and quantitative mass spectrometry approaches, and their abundance varies across treatments. To study these PTMs of FOXA1, we used CRISPR/Cas9 to create specific knock-in mutations to mimic or prevent acetylation events in MCF-7 cells. Specifically, we engineered MCF-7 cell lines where K270 was mutated to glutamine (K270Q) to mimic acetylation. And for comparison, we also created cell lines where K270 was mutated to arginine (K270R) to prevent acetylation of FOXA1. Our data, including FOXA1 ChIP-seq and RNA-seq, revealed the genomic redistribution of FOXA1 with these PTMs, which subsequently alters gene expression programs and promotes cell growth, migration, or chemoresistance. These results were confirmed in other ER+ cell lines (such as T47D cells) providing evidence for the generalizability of our findings. Taken together, our data suggest that inflammatory signaling signaling can reshape the enhancer landscape of FOXA1 through post-translational modifications, resulting in changes to estrogen signaling that have profound effects on breast cancer biology. Citation Format: Shen Li, Hector L. Franco, Hyunsoo Kim, Rosemary N. Plagens, Raul Mendez-Giraldez, Colby Tubbs, Venkat Malladi, Joseph Garay. Proinflammatory and estrogen signaling modulates the chemoresistance and metastasis of breast cancer cells through post-translational modifications of pioneering factor FOXA1 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-03.

LncRNA-BC069792 suppresses tumor progression by targeting KCNQ4 in breast cancer

BackgroundBreast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer. However, the specific mechanism remains not clear.MethodsIn this study, we explored the role of lncRNA BC069792 in breast cancer. In vitro and in vivo functional experiments were carried out in cell culture and mouse models. High-throughput next-generation sequencing technology and real-time fluorescence quantitative PCR technology were used to evaluate differentially expressed genes and mRNA expression, Western blot and immunohistochemical staining were used to detect protein expression. RNA immunoprecipitation assay and dual-luciferase activity assay were used to evaluate the competing endogenous RNAs (ceRNA), and rescue and mutation experiments were used for verification.ResultsWe found that lncRNA BC069792 was expressed at a low level in breast cancer tissues, and significantly decreased in breast cancer with high pathological grade, lymph node metastasis and high Ki-67 index groups. Moreover, BC069792 inhibited the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Mechanically, BC069792 acts as a molecular sponge to adsorb hsa-miR-658 and hsa-miR-4739, to up-regulate the protein expression of Potassium Voltage-Gated Channel Q4 (KCNQ4), inhibits the activities of JAK2 and p-AKT, and plays a role in inhibiting breast cancer growth.ConclusionsLncRNA BC069792 plays the role of tumor suppressor gene in breast cancer and is a new diagnostic index and therapeutic target in breast cancer.

Abstract P3-05-24: Correlation between the expression of JCAD in plasma exosomes and breast cancer

Abstract Background: Breast cancer is the most common cancer in women. The five-year survival rate of patients with stage 0 and stage I breast cancer is more than 95%, that of patients with stage II breast cancer is 80%, that of patients with stage III breast cancer is about 50%, and that of stage IV breast cancer is less than 30%. The stage of breast cancer is closely related to the prognosis. Exosomes are membrane vesicles secreted by living cells, which carry donor cell specific genetic information. They can exist stably in the circulatory system, so they are ideal biomarkers. Method: We collected 128 breast cancer patients (17 benign cases, 26 stage I cases, 36 stage II cases, 15 stage III cases and 34 stage IV cases) treated in our center from 2018 to 2019. We collected patients’ plasma and extracted exosomes, then we sent them for sequencing. We analyzed the difference between benign cases’ and stage IV cases’ sequencing results, and then we screen them by comprehensively considering the change trend of gene expression from benign cases to stage IV cases, the survival curve of patients and the lymph node metastasis. The clinicopathological factors and progression free survival (PFS) of breast cancer patients were analyzed by Cox regression. Finally, JCAD was selected. Using virus to stably overexpress JCAD and shRNA technology to knock down JCAD, the function of JCAD for the proliferation and metastasis of breast cancer was discussed through CCK8 cell proliferation experiment, EdU cell proliferation experiment, plate cloning experiment, transwell cell migration experiment. T-test and chi square test were performed on the data. P < 0.05 showed that the difference was statistically significant. Kaplan-Meier survival was used to analyze the long-term efficacy. Result 1. The expression of JCAD mRNA in exosomes increased from benign to stage 4 (P < 0.001). In addition, patients with lymph node metastasis had significantly higher expression of exosome JCAD (p=0.009), and the prognosis of patients with high expression of exosome JCAD was poor (p=0.003). This conclusion has also been verified in TCGA database. Finally, Cox regression analysis showed that JCAD was an independent prognostic factor affecting PFS (p=0.005). 2. JCAD is transmitted between cells in the form of exosomes: In breast cancer cells, stable transgenic strains with overexpression of JCAD and shRNA knockdown strains of JCAD were constructed. The exosomes were extracted by ultracentrifugation for RNA and protein level verification. It shows that there was a difference in the expression of JCAD in the exosomes of overexpression and knockdown strains. After adding exosome inhibitor GW4869 to culture cells, it was found that the level of JCAD mRNA secreted by exosomes decreased significantly. In addition, through PKH26 exosome fluorescence labeling method, it was found that JCAD was transmitted between cells in the form of exosomes. 3. JCAD can promote the proliferation and migration of breast cancer cells: This experiment verified the ability of JCAD to promote the proliferation of breast cancer cells through CCK8 cell proliferation experiment, EdU cell proliferation experiment, plate cloning experiment. Then the ability of JCAD to promote the migration of breast cancer cells was verified by transwell cell migration experiment. The exosomes were added to wild type cells for co-culture. It was further found that the exosomes with high expression of JCAD could promote the proliferation and migration of breast cancer cells. Conclusion JCAD is significantly overexpressed in stage IV patients and patients with lymph node metastasis. Moreover, JCAD is an independent prognostic factor affecting PFS, and the exosome JCAD is expected to become a potential prognostic liquid biopsy marker. Further cell experiments showed that JCAD can be transmitted between breast cancer cells through exosomes and promote the proliferation and metastasis of breast cancer cells. Citation Format: Douwaner Liu, Yayun Chi, Bingqiu Xiu, Qi Zhang, Wei-Ru Chi, Liyi Zhang, Min Xiong, Jiong Wu. Correlation between the expression of JCAD in plasma exosomes and breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-24.

Abstract P2-26-13: Gene deletion of insulin receptor in MCF-7L breast cancer cells by CRISPR gene editing

Abstract The type I insulin-like Growth Factor Receptor (IGF-IR) plays important roles in breast cancer cell proliferation and metastasis. However, preclinical and clinical research targeting IGF-IR also revealed the potential for insulin receptor (IR) to compensate for IGF-1R inhibition. IR is expressed as two isoforms (IRA and IRB) through alternative splicing. The IRB is expressed in adult tissues and thought responsible for cell metabolism while IRA is expressed in fetal development to enhance cell growth. Since breast cancer cells usually express both IGF-IR and IRA/IRB, differential signaling by IRA and IRB has not been clearly demonstrated. To further define the role for IR, we used CRISPR technology for gene editing in exon 2 of IR in MCF-7L cells to delete IR. Cells were selected for absent IR expression by flow cytometry and Sanger sequencing confirmed that one of the clones (CL35) had the targeted 4 base pair deletions in exon 2. Immunoblotting using an antibody to the  subunit of IR showed no expression in CL35. Unexpectedly, 10 nM insulin exposure resulted in downstream AKT and Erk1,2 phosphorylation. Immunoprecipitation showed that IGF-IR was activated by the insulin in CL35 cells. Insulin signaling in CL35 through IGF-IR was inhibited by huEM164, an anti IGF-IR monoclonal antibody. Compared to MCF-7L parental cells, CL35 was more sensitive to IGF-I and less sensitive to insulin as demonstrated by downstream AKT phosphorylation and MTT monolayer growth assay. Insulin stimulated monolayer growth of CL35 measured by MTT was not affected in testing serum free media (SFM) unless transferrin was deleted from the media. These data suggest IGF-IR can function to activate signaling in response to insulin. To ensure that our clone does not contain alternate splicing products resulting in aberrant IR proteins, we are further sequencing IR mRNA in CL35. In conclusion, we have established an IR null breast cancer cell line with reduced sensitivity to insulin and increased sensitivity to IGF-I. This cell line can be used to express the IRA and IRB isoforms to characterize their function in breast cancer cells. Citation Format: Xihong Zhang, Marvis Monteiro, Douglas Yee. Gene deletion of insulin receptor in MCF-7L breast cancer cells by CRISPR gene editing [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-13.

Asperolide A induces apoptosis and cell cycle arrest of human hepatoma cells with p53-Y220C mutant through p38 mediating phosphorylation of p53 (S33)

Asperolides A (AA), one of the new tetranorlabdane diterpenoids, is proved to inhibit the proliferation of lung cancer cells and bone metastasis of breast cancer cells. Herein, we report that AA induces apoptosis and cell cycle arrest of hepatoma cells. It intensely inhibits proliferation of Huh-7 cell, compared with HepG-2 and L02 cells. AA elevates the activity of mitogen-activated protein kinases (MAPKs), in which the activation of ERK and JNK improves cell survival. However, phosphorylation of p53 at S33 by p38 activation could be a principal factor in the AA-induced apoptosis and G2/M cell cycle arrest of Huh-7 cells. The S33 site of p53-Y220C mutant, as the specific activation site of p38, reactivates the wild-type function of mutant p53 protein, which leads to a higher sensitivity of Huh-7 cells to AA. These results provide new insights into the molecular mechanisms of AA as a developing mutant p53 rescue drug.

LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells.

EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (ΔZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (ΔZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells.

Sufentanil combined with parecoxib sodium inhibits proliferation and metastasis of HER2-positive breast cancer cells and regulates epithelial-mesenchymal transition.

Sufentanil combined with parecoxib sodium is a commonly used postoperative medication for cancer patients. However, the effects of this combination therapy on human epidermal growth factor receptor-2 (HER2)-positive breast cancer cells have still remained elusive. This study aimed to investigate the effects and potential mechanisms of sufentanil combined with parecoxib sodium on HER2-positive breast cancer cells. The cell counting kit-8 (CCK-8), colony formation, flow cytometry, scratch, transwell invasion, and angiogenesis assays were used to assess the proliferation, cell cycling, migration, invasion, and angiogenesis of HER2-positive breast cancer BT474 cells. Western blot assay was employed for detecting the expression levels of proteins involved in the cell cycle, migration, invasion, angiogenesis, and epithelial-mesenchymal transition (EMT). The in vivo effects of tumor growth and metastasis were examined by establishing an orthotopic transplantation mouse model of HER2-positive breast cancer (MMTV-PyMT). Functional assays indicated that sufentanil combined with parecoxib sodium induced blockade of HER2-positive breast cancer BT474 cells in the G1 phase of the cell cycle and inhibited cell proliferation, migration, angiogenesis, and invasion in vitro. Western blot assay revealed that sufentanil combined with parecoxib sodium downregulated the expression levels of cyclin D1, matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), vascular endothelial growth factor A (VEGFA), and EMT-related proteins (N-cadherin, Vimentin, and Snail), while up-regulated the expression level of E-cadherin in BT474 cells. In addition, it was found that sufentanil combined with parecoxib sodium inhibited tumor growth and metastasis in the orthotopic transplantation mouse model of HER2-positive breast cancer. Sufentanil combined with parecoxib sodium inhibited HER2-positive breast cancer progression, including cell proliferation, cell cycle, migration, invasion, and angiogenesis, and regulated EMT.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.