Metastasis Of Breast Cancer Cells Research Articles

Ethanolic extract of Euphorbia royleana Boiss. reduces metastasis of breast cancer cells and inhibits tumor progression in vivo.

Metastasis is the most devastating attribute of breast cancer (BC) that leads to high mortality. It is a complex process of tumor cell migration, invasion, and angiogenesis. In this study, we evaluated the effect of ERA on BC metastasis and BC progression in vivo. The transwell invasion/migration and wound healing assays showed that ERA treatment significantly reduced the invasion and migration of BC cell lines. The expression of mesenchymal (E-cadherin and N-cadherin), matrix metalloproteinases (MMP2, MMP9), and stemness markers (Oct3) were down-regulated by ERA. Furthermore, ERA down-regulated angiogenic chemokines (CXCL1/2/3, CXCL5, and CXCL12) expression in the highly metastatic MDA-MB-231 cell line. The clonogenic survival of BC cells was also reduced by ERA treatment. Strikingly, ERA prevented DMBA-induced tumor growth in Swiss albino mice as depicted by a high animal survival rate (84%) in the ERA group and histopathological analysis. Conclusively, this study revealed that ERA possesses anti-metastatic potential and also reduces the growth of BC in vivo. Moreover, the GC-MS data revealed the presence of biologically active compounds (Lupeol, Phytol, phytosterol) and some rare (9, 19-Cyclolanost) phyto metabolites in ERA extract. However, further studies are suggestive to identify and isolate the therapeutic agents from ERA to combat BC and metastasis.

Phytochemical identification and assessment of Amberboa ramosa Mediated Inhibition of Microtubule and EGFR Associated Growth and Metastasis of Breast Cancer Cells: In vitro and in silico perspective

Phytochemical identification and assessment of <i>Amberboa ramosa</i> Mediated Inhibition of Microtubule and EGFR Associated Growth and Metastasis of Breast Cancer Cells: In vitro and <i>in silico</i> perspective

Methyl CpG binding protein MBD2 has a regulatory role on the BRCA1 gene expression and its modulation by resveratrol in ER+, PR+ & triple-negative breast cancer cells

BackgroundResveratrol has demonstrated its ability to regulate BRCA1 gene expression in breast cancer cells, and previous studies have established the binding of MBD proteins to BRCA1 gene promoter regions. However, the molecular mechanism underlying these interactions remains to be elucidated. The aimed to evaluate the impact of MBD proteins on the regulation of BRCA1, BRCA2, and p16 genes and their consequential effects on breast cancer cells.MethodsEfficacy of resveratrol was assessed using the MTT assay. Binding interactions were investigated through EMSA, ChIP, & MeIP assay. Expression analyses of MBD genes and proteins were conducted using qRT-PCR and western blotting, respectively. Functional assays, including clonogenic, migratory, and sphere formation assays were used to assess cancer cells’ colony-forming, metastatic, and tumor-forming abilities. The cytotoxicity of resveratrol on cancer cells was also tested using an apoptosis assay.ResultsThe study determined an IC50 of 30µM for resveratrol. MBD proteins were found to bind to the BRCA1 gene promoter. Resveratrol exhibited regulatory effects on MBD gene expression, subsequently impacting BRCA1 gene expression and protein levels. Higher concentrations of resveratrol resulted in reduced colony and sphere formation, decreases migration of cancer cells, and an increases number of apoptotic cells in breast cancer cells.ImpactIdentification of MBD2-BRCA1 axis indicates their significant role in the induction of apoptosis and reduction of metastasis and proliferation in breast cancer cells. Further therapy can be designed to target these MBD proteins and resveratrol could be used along with other anticancer drugs to target breast cancer.ConclusionsIn conclusion MBD2 protein interact to the BRCA1 gene promoter, and resveratrol modulates MBD2 gene expression, which in turn regulates BRCA1 gene expression, and inhibits cell proliferation, migration, and induces apoptosis in ER+, PR+ & Triple negative breast cancer cells.

Berberine inhibits the progression of breast cancer by regulating METTL3-mediated m6A modification of FGF7 mRNA.

Berberine (BBR), an isoquinoline alkaloid from Coptidis rhizoma, has been found to have powerful activities against various human malignancies, including breast cancer. However, the underlying antitumor mechanisms of BBR in breast cancer remain poorly understood. Breast cancer cells were cultured and treated with different doses (0, 20, 40, and 60 μM) of BBR for 48 h. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. Fibroblast growth factor 7 (FGF7), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels and protein levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Interaction between METTL3 and FGF7 m6A was assessed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. Binding ability between IGF2BP3 and FGF7 mRNA was analyzed using RIP assay. BBR treatment hindered breast cancer cell proliferation, invasion, migration, and induced apoptosis. FGF7 expression was upregulated in breast cancer tissues, while its level was reduced in BBR-treated tumor cells. FGF7 upregulation relieved the repression of BBR on breast cancer cell malignant behaviors. In mechanism, METTL3 stabilized FGF7 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally improved FGF7 expression. BBR treatment inhibited breast cancer growth in vivo. BBR treatment blocked breast cancer cell growth and metastasis partly by regulating METTL3-mediated m6A modification of FGF7 mRNA, providing a promising therapeutic target for breast cancer treatment.

Abstract PO3-27-02: A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells

Abstract Background. PTK6/Brk, a non-receptor tyrosine kinase, is an oncogenic driver for several tumor types, including breast, prostate, liver, and pancreatic cancers. PTK6 is expressed in all breast cancer subtypes and higher levels of expression are associated with worse patient outcomes. The optimal approach to targeting PTK6 for therapeutic benefit has not been established. Although several inhibitors of PTK6 kinase activity have been developed, none have thus far been successfully translated to clinical trials. This may be in part due to kinase activity-independent functions of PTK6, for which there is growing evidence, that are not effectively targeted by traditional small molecule kinase inhibitors. Downregulation of PTK6 using shRNA inhibits growth, invasion/migration, survival and metastasis of breast cancer cells, including those that are resistant to standard therapies. PTK6 kinase inhibitor treatment phenocopies effects of PTK6 downregulation with respect to some, but not all PTK6-driven oncogenic activities. Moreover, growth of PTK6 CRISPR-targeted cells can be rescued either with wild type or catalytically inactive PTK6, supporting kinase activity-independent activities of PTK6 in regulating growth and viability of breast cancer cells. Therefore, PROTAC degraders may be a better way to chemically target and downregulate PTK6 expression in cancer, thereby phenocopying the effects of PTK6 shRNA. Methods. We developed a first-in-class PTK6 degrader using PROTAC (proteolysis-targeting chimeric) technology to target PTK6 expression in breast cancer cells of diverse subtypes. Degrader controls were also synthesized to assess specificity of MS105’s effects. Results. PTK6 degrader, but not degrader controls, potently downregulates PTK6 expression in multiple breast cancer cell types. Proteomic and kinome profiling confirm the specificity of our PTK6 degrader. PTK6 PROTAC degrader inhibits viability and induces apoptosis of PTK6-expressing breast cancer cells, whereas treatment with parent kinase inhibitor or degrader controls that do not engage PTK6 or E3 ligase have minimal effect. Treatment with PTK6 degrader, but not kinase inhibitor or degrader controls, induces expression of pro-apoptotic Bim, phenocopying the effects observed with PTK6 shRNA. In contrast, PTK6 degrader and kinase inhibitor are comparably effective in inhibiting cell migration, supporting the differential kinase dependency of PTK6-dependent oncogenic functions. Therefore, our PTK6 PROTAC degrader effectively inhibits both kinase-dependent and independent oncogenic activities of PTK6. Our studies support the development of PTK6 PROTAC degraders as a preferred approach to clinically targeting PTK6 in cancer. Citation Format: Hanna Irie, Criseyda Martinez, Yan Xiong, Alison Bartkowski, Jessica Byerly, Jian Jin. A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-27-02.

Abstract PO2-24-09: Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype

Abstract Aidan Moriarty Mentor: Dr. Min Yu Hypoxic Memory in Breast Cancer Cells Metastasis is the main cause of tumor-related mortality in breast cancer patients. Improving our understanding of the metastatic cascade is imperative to improve patient outcomes. Prior research indicates cancer cells with increased metastatic potential have acquired adaptive properties induced by factors in the tumor microenvironment (TME), including hypoxia. However, previous hypoxia research has been primarily focused on signaling changes that occur when cancer cells are in the hypoxic TME and does not account for long-term changes that may occur once cancer cells re-enter normoxia, such as when they metastasize. Our research in patient-derived circulating tumor cell (CTCs) lines and breast cancer cell lines has indicated that certain transcriptional changes are maintained after cells leave hypoxia, and that these post-hypoxic CTCs maintain a higher metastatic potential compared to cells not exposed to hypoxia. Taking an un-biased approach to define changes in hypoxia-exposed breast cancer cells upon reoxygenation, we performed bulk RNA-Seq on luminal breast cancer cell lines in various oxygen conditions over time. We found that a subset of genes that were differentially expressed in hypoxic conditions were maintained as up- or down-regulated after cells were reoxygenated, indicating a “hypoxic memory”. Additionally, a subset of genes were differentially expressed in the post-hypoxic cells compared to normoxia controls, demonstrating novel and prolonged gene expression changes induced by hypoxia exposure. We performed functional assays and observed that this post-hypoxic state is associated with a more aggressive phenotype in vitro. Current research in our lab is on-going to understand the epigenetic changes that may be responsible for this hypoxic memory to further understand the consequences of hypoxic memory in CTC biology and metastasis. Finally, we have designed a dual-reporter hypoxia-tracing system to validate our findings in vivo. This research will improve our understanding of the prolonged influence of hypoxia on metastatic potential in breast cancer cells and has the potential to inform future therapeutic strategies. Citation Format: Aidan Moriarty, Oihana Iriondo, Desirae Mecenas, Yilin Li, Yonatan Amzaleg, Remi Klotz, Min Yu. Prolonged Effects of Hypoxia in Luminal Breast Cancer Cells Promotes an Aggressive Phenotype [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-09.

A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis

Breast cancer (BC) poses a great threat to women’s health. Neuronal regeneration related protein (NREP) is a multifunctional protein that is involved in embryonic development, regeneration, and human disease. However, the biological function of NREP in tumors is rarely reported and its role in BC remains unknown. Bioinformatics analysis showed that NREP is highly expressed and closely correlated with poor survival in BC patients. Under hypoxic conditions, NREP was upregulated in BC cells, and this promotion was reversed by hypoxia-inducible factor HIF-1α suppression. Luciferase reporter system and chromatin immunoprecipitation assays confirmed that HIF-1α directly binds to the promoter of NREP to increase the transcriptional activity of NREP. NREP suppression inhibited cell proliferation, arrested the cell cycle at the G1/S phase, and promoted apoptosis and caspase-3 activity in BC cells. Suppression of NREP decreased the tube formation ability of HUVECs. In addition, NREP downregulation showed an inhibition effect on cell migration, invasion, and EMT of BC cells. In NREP overexpressed cells, all these changes were reversed. In vivo, animal experiments also confirmed that NREP promotes BC tumor growth and metastasis. In addition, NREP promoted cellular glycolysis and enhanced the levels of glucose consumption, ATP, lactate production, and glucose transporters expression in NREP-overexpressed BC cells. In summary, our results demonstrated that NREP could be transcriptional activated by HIF-1α, which may aggravate BC tumor growth and metastasis by promoting cellular glycolysis. This result suggested that NREP may play an essential part in BC progression.

CircLIFR-007 reduces liver metastasis via promoting hnRNPA1 nuclear export and YAP phosphorylation in breast cancer

Cancer metastasis is the major cause of death in patients with breast cancer (BC). The liver is a common site of breast cancer metastasis, and the 5-year survival rate of patients with breast cancer liver metastases (BCLMs) is only about 8.5 %. CircRNAs are involved in a variety of cancer-related pathological behaviors, and their unique structure and resistance to RNA degradation enable them to serve as ideal diagnostic biomarkers and therapeutic targets. Therefore, it is important to investigate the role and molecular mechanism of circRNAs in cancer metastasis. CircLIFR-007 was identified as a critical circular RNA in BC metastasis by circRNAs microarray and qRT-PCR experiment. Cell function assays were performed to explore the effect of circLIFR-007 in breast cancer cells. Experiments in vivo validated the function of circLIFR-007. Several molecular assays were performed to investigate the underlying mechanisms. We found that circLIFR-007 acted as a negative controller in breast cancer liver metastasis. CircLIFR-007 upregulates the phosphorylation level of YAP by exporting hnRNPA1 to promote the combination between hnRNPA1 and YAP in the cytoplasm. Overexpression of circLIFR-007 suppressed the expression of liver metastasis-related proteins, SREBF1 and SNAI1, which were regulated by transcription factor YAP. Functionally, circLIFR-007 inhibits the proliferation and metastasis of breast cancer cells both in vivo and in vitro.

LIFU/MMP-2 dual-responsive release of repurposed drug disulfiram from nanodroplets for inhibiting vasculogenic mimicry and lung metastasis in triple-negative breast cancer

BackgroundVasculogenic mimicry (VM), when microvascular channels are formed by cancer cells independent of endothelial cells, often occurs in deep hypoxic areas of tumors and contributes to the aggressiveness and metastasis of triple-negative breast cancer (TNBC) cells. However, well-developed VM inhibitors exhibit inadequate efficacy due to their low drug utilization rate and limited deep penetration. Thus, a cost-effective VM inhibition strategy needs to be designed for TNBC treatment.ResultsHerein, we designed a low-intensity focused ultrasound (LIFU) and matrix metalloproteinase-2 (MMP-2) dual-responsive nanoplatform termed PFP@PDM-PEG for the cost-effective and efficient utilization of the drug disulfiram (DSF) as a VM inhibitor. The PFP@PDM-PEG nanodroplets effectively penetrated tumors and exhibited substantial accumulation facilitated by PEG deshielding in a LIFU-mediated and MMP-2-sensitive manner. Furthermore, upon exposure to LIFU irradiation, DSF was released controllably under ultrasound imaging guidance. This secure and controllable dual-response DSF delivery platform reduced VM formation by inhibiting COL1/pro-MMP-2 activity, thereby significantly inhibiting tumor progression and metastasis.ConclusionsConsidering the safety of the raw materials, controlled treatment process, and reliable repurposing of DSF, this dual-responsive nanoplatform represents a novel and effective VM-based therapeutic strategy for TNBC in clinical settings.Graphical

GATA4 regulates the transcription of MMP9 to suppress the invasion and migration of breast cancer cells via HDAC1-mediated p65 deacetylation

GATA-binding protein 4 (GATA4) is recognized for its significant roles in embryogenesis and various cancers. Through bioinformatics and clinical data, it appears that GATA4 plays a role in breast cancer development. Yet, the specific roles and mechanisms of GATA4 in breast cancer progression remain elusive. In this study, we identify GATA4 as a tumor suppressor in the invasion and migration of breast cancer. Functionally, GATA4 significantly reduces the transcription of MMP9. On a mechanistic level, GATA4 diminishes MMP9 transcription by interacting with p65 at the NF-κB binding site on the MMP9 promoter. Additionally, GATA4 promotes the recruitment of HDAC1, amplifying the bond between p65 and HDAC1. This leads to decreased acetylation of p65, thus inhibiting p65’s transcriptional activity on the MMP9 promoter. Moreover, GATA4 hampers the metastasis of breast cancer in vivo mouse model. In summary, our research unveils a novel mechanism wherein GATA4 curtails breast cancer cell metastasis by downregulating MMP9 expression, suggesting a potential therapeutic avenue for breast cancer metastasis.

Multiwalled Carbon Nanotubes-Reprogrammed Macrophages Facilitate Breast Cancer Metastasis via NBR2/TBX1 Axis.

In recent years, carbon nanotubes have emerged as a widely used nanomaterial, but their human exposure has become a significant concern. In our former study, we reported that pulmonary exposure of multiwalled carbon nanotubes (MWCNTs) promoted tumor metastasis of breast cancer; macrophages were key effectors of MWCNTs and contributed to the metastasis-promoting procedure in breast cancer, but the underlying molecular mechanisms remain to be explored. As a follow-up study, we herein demonstrated that MWCNT exposure in breast cancer cells and macrophage coculture systems promoted metastasis of breast cancer cells both in vitro and in vivo; macrophages were skewed into M2 polarization by MWCNT exposure. LncRNA NBR2 was screened out to be significantly decreased in MWCNTs-stimulated macrophages through RNA-seq; depletion of NBR2 led to the acquisition of M2 phenotypes in macrophages by activating multiple M2-related pathways. Specifically, NBR2 was found to positively regulate the downstream gene TBX1 through H3k27ac activation. TBX1 silence rescued NBR2-induced impairment of M2 polarization in IL-4 & IL-13-stimulated macrophages. Moreover, NBR2 overexpression mitigated the enhancing effects of MWCNT-exposed macrophages on breast cancer metastasis. This study uncovered the molecular mechanisms underlying breast cancer metastasis induced by MWCNT exposure.

[Retracted] Upregulation of microRNA‑181b inhibits CCL18‑induced breast cancer cell metastasis and invasion via the NF‑κB signaling pathway.

[This retracts the article DOI: 10.3892/ol.2016.5230.].

Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis

Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.

Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

Novel components in the noncanonical Hippo pathway that mediate the growth, metastasis, and drug resistance of breast cancer (BC) cells need to be identified. Here, we showed that SAM and SH3 domain containing protein 1 (SASH1) expression is negatively correlated with mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) expression in a subpopulation of patients with luminal-subtype BC. Downregulated SASH1 and upregulated MAP4K4 synergistically regulated the proliferation, migration, and invasion of luminal-subtype BC cells. The expression of LATS2, SASH1 and YAP1 and the phosphorylation of YAP1 were negatively regulated by MAP4K4, and LATS2 then phosphorylated SASH1 to form a novel MAP4K4-LATS2-SASH1-YAP1 cascade. Dephosphorylation of Yes1 associated transcriptional regulator (YAP1), YAP1/TAZ nuclear translocation and downstream transcriptional regulation of YAP1 were promoted by the combined effects of ectopic MAP4K4 expression and SASH1 silencing. Targeted inhibition of MAP4K4 blocked proliferation, cell migration and ER signaling both in vitro and in vivo. Our findings reveal a novel MAP4K4-LATS2-SASH1-YAP1 phosphorylation cascade, a noncanonical Hippo pathway that mediates ER signaling, tumorigenesis and metastasis in breast cancer. Targeted intervention with this noncanonical Hippo pathway may constitute a novel alternative therapeutic approach for endocrine-resistant BC.

Decreased LDHB expression in breast tumor cells causes NK cell activation and promotes tumor progression.

Abnormal metabolism is the underlying reason for breast cancer progression. Decreased lactate dehydrogenase B (LDHB) has been detected in breast cancer but the function of LDHB remains unknown. Western blot was used to analyze LDHB expression in breast cancer cells. The impact of LDHB on tumor cell migration and invasion was determined using Transwell assays, wound healing assays, and a mouse lung metastasis model. Subcutaneous tumor formation, a natural killer (NK) cell cytotoxicity assay, and flow cytometry evaluated NK cell activation. Immunofluorescence and quantitative real-time PCR detected NK cell activation markers. Kaplan-Meier analysis evaluated the effect of immune cell infiltration on prognosis. Single-sample gene set enrichment analysis determined NK cell activation scores. A support vector machine predicted the role of LDHB in NK cell activation. In this study we showed that LDHB inhibits the breast cancer cell metastasis and orchestrates metabolic reprogramming within tumor cells. Our results revealed that LDHB-mediated lactic acid clearance in breast cancer cells triggers NK cell activation within the tumor microenvironment. Our findings, which were confirmed in a murine model, demonstrated that LDHB in tumor cells promotes NK cell activation and ultimately results in the eradication of malignant cells. Clinically, our study further validated that LDHB affects immune cell infiltration and function. Specifically, its expression has been linked to enhanced NK cell-mediated cytotoxicity and improved patient survival. Furthermore, we identified LDHB expression in tumors as an important predictor of NK cell activation, with strong predictive ability in some cancers. Our results suggest that LDHB is a promising target for activating the tumor immune microenvironment in breast cancer, where LDHB-associated lactic acid clearance leads to increased NK cell activity. This study highlights the critical role of LDHB in regulating immune responses and its potential as a therapeutic target for breast cancer.

Abstract 4122: Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis

Abstract INTRODUCTION: Metastasis is the major cause of cancer-related deaths (90%). Intercellular communication between cancer cells and with cells in their environment plays a significant function in tumor progression. In fact, secreted factors like cytokines and extracellular vesicles (EVs) can either halt or stimulate tumorigenesis as well as metastasis. We have previously reported, that the integrin B3 (ITGB3) is required for metastasis of breast cancer cells in vivo (Sese et al. Oncotarget, 2017). We furthermore demonstrated, that EVs mediated colony growth is dependent on ITGB3 and that endocytosis mediated uptake of EVs relies on the interaction of ITGB3 with integrin av (ITGav) and heparan sulphated glycoproteins (HSPGs) (Fuentes et al. Nat. Commun., 2021). OBJECTIVE: The objective of this work is focused on identifying inhibitors targeting the ITGav/B3/HSPGs complex to prevent extracellular vesicle uptake in cancer cells and to test their ability to prevent metastasis. METHODOLOGY. Inhibitors targeting ITGB3/ITGAV or HSPGs were either commercially available or obtained through collaborations and single chain antibodies (scFv) were produced in house. Inhibition of fluorescent labeled EVs was determined by FACS analysis or Incucyte based measurements and the efficacy of inhibitors was determined in a dose dependent manner. Toxicity of inhibitors was studied by crystal violet-based assay in normal and tumor cell lines. Tail vein injection of luciferase labeled MDA-MB-231 cells into nude mice was used as model to study metastatic diseases under treatment with the identified inhibitors. Metastasis formation was monitored by bioluminescence imaging and lungs were examined pathologically at end point. Overall survival was determined. RESULTS: Among the tested Integrin and HSPGs targeting compounds, we identified an integrin-binding antibody, an integrin binding scFv, an RGD-mimetic compound and an HSPGs-binding peptide as inhibitors of EV uptake. Inhibition was validated in a dose dependent manner and toxicity studies did not show adverse effects. Three of those inhibitors, targeting either ITGB3/ITGAV or HSPGs were tested in mice models in vivo and all inhibitors could significantly reduce the formation of metastasis and strongly increase the overall survival. CONCLUSION: We have identified four drug candidates that target the ITGav/B3/HSPGs complex and are able to prevent EV uptake, demonstrating that EV uptake is a druggable process. Even more, the inhibitors tested in vivo could strongly reduce metastasis formation and increase overall survival. This data supports our hypothesis, that targeting EV mediated intercellular communication is a valuable approach to combat cancer metastasis. Citation Format: Elena Muro-Blanc, Rocío Bayona-Ramón y Cajal, Marta Cano-Galietero, Sara García-Ortega, Chiara Falciani, Luisa Bracci, Alberto J. Schuhmacher, Stefan Hümmer, Santiago Ramón y Cajal. Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4122.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

Abstract 4291: Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models

Abstract The adhesion G-protein coupled- receptor 1 ADGRB1/Bai1 was identified as a phosphatidylserine receptor that cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. This function of Bai1 was also shown to promote myoblasts fusion. Cell-cell fusion has been demonstrated to occur in normal physiological functions as well as in diseases. Studies have shown in cancer that cell-cell fusion increases tumor heterogeneity, drug resistance, and metastasis as hybrids acquire new properties that allow them to migrate and proliferate at new sites. Activation of ELMO-Dock signaling pathway has been associated with breast cancer metastasis. Moreover, ELMO1 has been identified as a modifier of breast cancer risk for BRCA mutation carriers. Rac1 was also shown to be overexpressed in proliferative breast disease, preinvasive and invasive breast carcinoma as well as lymph node metastases. Our recent studies showed that breast cancer cell fusion with bone marrow-derived mesenchymal stem cells was modulated by hypoxic conditions and promoted by apoptosis. Our hypothesis is that hypoxia stress induced apoptosis activates the phosphatidylserine receptor Bai1, and it signals through the ELMO/DOCK180/Rac to promote breast cancer cell fusion-driven tumor heterogeneity and metastasis. In this study, we determined the ability of Bai1 depleted breast cancer cells to induce tumor growth in immunocompromised mice. We used Bai1 sgRNA to inhibit Bai1 expression in the breast cancer cell MDA-MB-231. These cells (1x 106) together with bone marrow-derived mesenchymal stem cells (MSCs) (1x 106) were injected into the fat pad of the breast of 15 NOD SCID female mice. Control mice (15) received the same cells with no inhibition of Bai1. The mice were monitored for eight weeks. We found that Bai1 inhibition significantly reduced tumor growth in these mice in the course of time (P&amp;lt;0.05). The average tumor weight was also significantly reduced when Bai1 was inhibited (P&amp;lt;0.001). RNA sequencing of tumors generated from these mice revealed a significant increase in the expression of genes driving ferroptosis and a reduction in critical oncogenes in the experiment group compared to the control group. These findings will be helpful in the development of new classes of drugs effective in breast cancer treatment. Grant Support The NIH/NIMHD grant 5U54MD015929-03, the Society for Investigative Dermatology (SID) Freinkel Diversity Fellowship, and NSF Award Notice for Award ID 2142465 (awarded to FNK). Citation Format: Felicite Kamdem Noubissi, Oluwatowin Odubanjo, Hung-Chung Huang, Brenda Ogle, Paul Tchounwou. Bai1 inhibition promotes ferroptosis of breast cancer cells to prevent tumor growth in human xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4291.

LSD1 modulates the bone metastasis of breast cancer cells through hnRNPA2B1-mediated sorting of exosomal miRNAs

Bone metastasis is a key contributor to morbidity and mortality of breast cancer patients. We have previously shown that exosomal miRNAs derived from LSD1 knockdown (KD) breast cancer cells inhibit osteoblast differentiation and promote osteoclast differentiation. However, how LSD1 regulates exosomal miRNAs and whether miRNAs promote bone metastasis through the formation of pre-metastatic niches remains unclear. In vivo experiments demonstrates that exosomes derived from LSD1 KD breast cancer cells significantly promoted bone metastasis. To explore the mechanism underlying the effect of LSD1 on exosomes in breast cancer cells, exosomal and cellular miRNAs from control, LSD1 KD, and rescue cells were sequenced. Interestingly, approximately 80% of LSD1-associated miRNAs were downregulated in exosomes from LSD1 KD cells. The consensus sequence UAGGGC, was identified in many miRNAs downregulated in LSD1 KD exosomes. We found that hnRNPA2B1 regulated the exosomal sorting of miR-6881-3p and some other miRNAs. LSD1 deficiency reduced hnRNPA2B1 expression in breast cancer cells by decreasing the level of H3K9me2 demethylation in the promoter region of the hnRNPA2B1 gene. Our study revealed that LSD1 plays a crucial role in the regulation of exosomal sorting of miRNA.