scholarly journals Loss of Neuroligin 4X Induces An Intrinsic Innate Immune Response in TNBC

2021 ◽  
Author(s):  
Alehegne Yirsaw ◽  
Muhammad G Omar ◽  
Isra A Elhussin ◽  
Dequarius King ◽  
Henry J Henderson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Adhesion ◽  
Cancer Cells ◽  
Immune Checkpoint ◽  
Breast Cancer Cells ◽  
Innate Immune ◽  
Rna Seq ◽  
Interferon Signaling ◽  
Tnbc Patient

Abstract Background Immune checkpoint blockade therapies, which act on T cell inhibitory receptors, including CTLA-4 and PD-1, induce durable responses across diverse cancers. However, most patients do not respond to these therapies, and initially responsive cancers may relapse. Identifying molecular mechanisms that influence therapeutic responses and resistance is critical to realize the full therapeutic potential of immune checkpoint inhibitors. The presence of immune infiltrates in the tumor microenvironment is associated with positive outcomes in breast cancer, specifically in triple-negative breast cancer (TNBC). The underlying mechanisms driving this response are unclear. We have previously identified Neuroligin 4X (NLGN4X) as a protein expressed in TNBC.Methods Bioinformatic analysis was used for pathway analysis of TCGA TNBC patient dataset. Immunohistochemistry was performed on breast cancer tissue microarray for NLGN4X protein expression. RNA-seq was performed on MDA-MB-231 breast cancer cells for differential gene expression upon gene knockdown. Cytokine array, western blot, cell adhesion array and Nanostring was performed to determine the role of NLGN4X in TNBC.Results In this study, we report that NLGN4X expression is lost in breast cancer with lymph node metastasis. Its expression negatively correlates with immune markers in vitro, The Cancer Genome Atlas (TCGA) TNBC patient dataset, and metastatic breast cancer tissues. RNA-seq analysis of the MDA-MB-231 breast cancer cell line, silenced for NLGN4X by siRNA showed more than 500 differentially regulated genes. GSEA analysis of these genes revealed upregulation of interferon signaling pathway, cytokine signaling, and downregulation of cholesterol metabolism and lipid metabolism pathways. NLGN4X knockdown induced loss of cell adhesion, epithelial to mesenchymal transition (EMT), and MAVS-IRF7 signaling in breast cancer cells. Interestingly, analysis of the TCGA dataset of 104 TNBC patients also showed interferon signaling (IFN) as one of the significant pathways downregulated in TNBC patients expressing NLGN4X.Conclusion Loss of NLGN4X leads to innate immune activation in breast cancer and coincides with an aggressive phenotype of cancer. This study identifies the role of NLGN4X in regulating interferon signaling and immune microenvironment in TNBC.

scholarly journals Loss of Neuroligin 4X Induces an Intrinsic Innate Immune Response in TNBC

2021 ◽  
Author(s):  
Alehegne Yirsaw ◽  
Muhammad G Omar ◽  
Isra A Elhussin ◽  
Dequarius King ◽  
Henry J Henderson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Adhesion ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Innate Immune ◽  
Rna Seq ◽  
Interferon Signaling ◽  
Mesenchymal Transition ◽  
Tnbc Patient

Abstract BackgroundImmune checkpoint blockade therapies, which act on T cell inhibitory receptors, including CTLA-4 and PD-1, induce durable responses across diverse cancers. However, most patients do not respond to these therapies, and initially responsive cancers may relapse. Identifying molecular mechanisms that influence therapeutic responses and resistance is critical to realize the full therapeutic potential of immune checkpoint inhibitors. The presence of immune infiltrates in the tumor microenvironment is associated with positive outcomes in breast cancer, specifically in triple-negative breast cancer (TNBC). The underlying mechanisms driving this response are unclear. We have previously identified Neuroligin 4X (NLGN4X) as a protein expressed in TNBC.MethodsBioinformatic analysis was used for pathway analysis of TCGA TNBC patient dataset. Immunohistochemistry was performed on breast cancer tissue microarray for NLGN4X protein expression. RNA-seq was performed on MDA-MB-231 breast cancer cells for differential gene expression upon gene knockdown. Cytokine array, western blot, cell adhesion array and Nanostring was performed to determine the role of NLGN4X in TNBC.ResultsIn this study, we report that NLGN4X expression is lost in breast cancer with lymph node metastasis. Its expression negatively correlates with immune markers in vitro, The Cancer Genome Atlas (TCGA) TNBC patient dataset, and metastatic breast cancer tissues. RNA-seq analysis of the MDA-MB-231 breast cancer cell line, silenced for NLGN4X by siRNA showed more than 500 differentially regulated genes. GSEA analysis of these genes revealed upregulation of interferon signaling pathway, cytokine signaling, and downregulation of cholesterol metabolism and lipid metabolism pathways. NLGN4X knockdown induced loss of cell adhesion, epithelial to mesenchymal transition (EMT), and MAVS-IRF7 signaling in breast cancer cells. Interestingly, analysis of the TCGA dataset of 104 TNBC patients also showed interferon signaling (IFN) as one of the significant pathways downregulated in TNBC patients expressing NLGN4X.ConclusionLoss of NLGN4X leads to innate immune activation in breast cancer and coincides with an aggressive phenotype of cancer. This study identifies the role of NLGN4X in regulating interferon signaling and immune microenvironment in TNBC.

scholarly journals Role of the Protein Tyrosine Kinase Syk in Regulating Cell-Cell Adhesion and Motility in Breast Cancer Cells

2009 ◽  
Vol 7 (5) ◽  
pp. 634-644 ◽  
Author(s):  
Xiaoying Zhang ◽  
Ulka Shrikhande ◽  
Bethany M. Alicie ◽  
Qing Zhou ◽  
Robert L. Geahlen
Keyword(s):  
Breast Cancer ◽  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine ◽  
Cell Cell

scholarly journals 931 LncRNA Malat1 regulated Serpinb6b signaling suppresses pyroptosis and governs breast cancer dormancy reactivation and through immune evasion

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A977-A977
Author(s):  
Dhiraj Kumar ◽  
Filippo Giancotti
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Single Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Immune Evasion ◽  
Breast Cancer Cells ◽  
Rna Seq

BackgroundMetastatic relapse is the major causes of mortality in patients with cancer and occur due to metastatic reactivation of dormant tumor cells. Early dissemination of tumor cells undergoing a protected period of dormancy in the target organs potentially explains this prevalent clinical behavior.1–4 Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 is one of the most abundant and highly conserved nuclear lncRNAs and have shown the associated with metastasis and serving as a predictive marker for various tumor progression.5 However, the correlation of tumor intrinsic lncRNAs in regulation of tumor dormancy and immune evasion is largely unknown.MethodsUsing an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation.4 To dissect the functional role of Malat1 in metastatic reactivation, we developed a clean Malat1 knockout (KO) model using paired gRNA CRISPR-Cas9 in metastatic murine syngeneic breast cancer. As proof of concept we also used inducible knockdown system under in vivo models. To delineate the immune microenvironment, we used single cell RNA-seq, ChIRP-seq, multicolor flowcytometry, RNA-FISH, and coculture experiments.ResultsOur data revealed that deletion of Malat1 induces dormancy and attenuated the metastatic colonization resulting in long-term survival of syngeneic mice model. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Interestingly, 4T1-Malat1 KO dormant breast cancer cells exhibit metastatic outgrowth in T cells defective mice. Our single-cell RNA-seq and multicolor flowcytometry evaluation reveal enhanced T cells and reduced neutrophils proportions in mice with Malat1 KO cells. This indicates a critical role of immune microenvironment via Malat1-dependent immune evasion. Additionally, Malat1 KO inhibits cancer stemness properties. Similarly, RNA-seq and ChIRP-seq data suggest that KO of Malat1 hampers immune evasion and downregulates metastasis associated genes including Serpins and Wnts. Additionally, our data strongly suggests that Malat1 KO cells persists as non-proliferative dormant cells in lung due to CD8+ T cell-umpired immune activity. Interestingly, rescue experiments suggest that Malat1 or Serpinb6b protects T cell-induced cell death and induces dormancy re-awakening thereby rescue the metastatic potential of 4T1 Malat1 KO cells. Combination of Malat1 ASO with double immune checkpoint inhibitors greatly affects the metastatic outgrowth in breast cancer.ConclusionsTaken together, our studies demonstrate that tumor intrinsic Malat1 regulates Serpinb6b that eventually controls immune evasion and promote dormancy metastatic reactivation.AcknowledgementsNGS data generated was supported by Core grant CA016672(ATGC) and NIH 1S10OD024977-01 award to the ATGC. Single cell RNA sequencing data was supported by the CPRIT Single Core grant RP180684. The Advanced Cytometry & Sorting Core Facility is supported by NCI P30CA016672.ReferencesArun G, Diermeier S, Akerman M, et al. Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev 2016 January 1;30(1):34–51.Filippo G Giancotti. Mechanisms governing metastatic dormancy and reactivation. Cell 2013 November 7;155(4):750–764.Gao H, Chakraborty G, Lee-Lim AP, et al. The BMP inhibitor Coco reactivates breast cancer cells at lung metastatic sites. Cell 2012b;150:764–779.Gao H, Chakraborty G, Lee-Lim AP, et al. Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation. Proc Natl Acad Sci U S A 2014 November 18;111(46):16532–16537.Huang D, Chen J, Yang L, et al. NKILA lncRNA promotes tumor immune evasion by sensitizing T cells to activation-induced cell death. Nat Immunol 2018;19:1112–1125.

scholarly journals Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor

2021 ◽  
Vol 13 (1) ◽  
pp. 17-29
Author(s):  
Emann M Rabie ◽  
Sherry X Zhang ◽  
Andreas P Kourouklis ◽  
A Nihan Kilinc ◽  
Allison K Simi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Type I ◽  
Matrix Degradation ◽  
Human Breast ◽  
Rate Of Invasion

Abstract Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

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