Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary ...epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)-directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell-cell contacts and persistent activation of Prkd1. In patients with breast cancer,
and
expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally,
knockdown
blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination. SIGNIFICANCE: Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/2/204/F1.large.jpg.
Abstract
BACKGROUND: Metastatic disease drives breast cancer mortality. We recently discovered that leading cells at the invasive edge of mammary tumor organoids retain a conserved basal epithelial ...program defined by their expression of keratin-14 (K14), establishing K14 as a good marker of invasive breast cancer cells. K14-positive invasive cells also exhibit characteristics that make them targets of immunosurveillance by natural killer (NK) cells. While NK cells are key immune mediators in the control of metastasis, our understanding of the specific mechanisms behind this regulation and its eventual evasion by metastatic cells remains incomplete. METHODS: We have developed a novel preclinical 3D co-culture assay to discover mechanisms behind interactions between K14+ invasive breast cancer cells and NK cells. Combined with in vivo assays of metastasis, we are able to determine how NK cells limit the early stages of metastasis and also how tumor cells can influence key NK cell properties. RESULTS: In ex vivo co-culture assays of NK cells isolated from healthy mouse donors and mammary tumor organoids from MMTV-PyMT and C31T mouse models of breast cancer, we demonstrate that NK cells limit the early stages of metastasis, namely invasion and colony formation. Antibodies to invasive K14+ cells were able to enhance the ability of NK cells to limit colony formation, suggesting antibody-dependent cell mediated cytotoxicity. Surprisingly, when isolated from tumor bearing mice, NK cells did not limit invasion and instead promoted colony formation. The in vivo adoptive transfer of NK cells from healthy donors prevents the progression of early lung metastatic seeds to macrometastases, while the adoptive transfer of cells isolated from tumor bearing donors promotes macrometastatic development. This growth promoting phenotype can be neutralized with antibodies targeting inhibitory cell surface receptors. CONCLUSIONS: Our ex vivo and in vivo data demonstrate that healthy donor NK cells can limit metastasis through the directed cytotoxicity against pioneering K14+ invasive cells. However, prolonged exposure to tumors reprogram NK cells from tumor killing to tumor promoting, specifically in promoting the outgrowth of macrometastases. Further, we can neutralize this effect using NK cell specific inhibitory antibodies. This is the first time inhibitory signaling on NK cells have been linked with a growth promoting phenotype. These data can provide insight into when the use of NK cell directed therapies can be used to treat or prevent clinically relevant metastatic disease.
Citation Format: Isaac S. Chan, Hildur Knútsdoóttir, Gayathri Ramakrishnan, Veena Padmanaban, Joel S. Bader, Elizabeth M. Jaffee, Andrew J. Ewald. A dual role of natural killer cells in breast cancer metastasis abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-01.
Abstract
Natural killer (NK) cells have potent antitumor and antimetastatic activity. It is incompletely understood how cancer cells escape NK cell surveillance. Using ex vivo and in vivo models of ...metastasis, we establish that keratin-14+ breast cancer cells are vulnerable to NK cells. We then discovered that exposure to cancer cells causes NK cells to lose their cytotoxic ability and promote metastatic outgrowth. Gene expression comparisons revealed that healthy NK cells have an active NK cell molecular phenotype, whereas tumor-exposed (teNK) cells resemble resting NK cells. Receptor–ligand analysis between teNK cells and tumor cells revealed multiple potential targets. We next showed that treatment with antibodies targeting TIGIT, antibodies targeting KLRG1, or small-molecule inhibitors of DNA methyltransferases (DMNT) each reduced colony formation. Combinations of DNMT inhibitors with anti-TIGIT or anti-KLRG1 antibodies further reduced metastatic potential. We propose that NK-directed therapies targeting these pathways would be effective in the adjuvant setting to prevent metastatic recurrence.
Citation Format: Isaac S. Chan, Hildur Knútsdóttir, Gayathri Ramakrishnan, Veena Padmanaban, Manisha Warrier, Juan Carlos Ramirez, Matthew Dunworth, Hao Zhang, Elizabeth M. Jaffee, Joel S. Bader, Andrew J. Ewald. Cancer cells educate natural killer cells to a metastasis-promoting cell state abstract. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO039.
Gouty arthritis is characterized by chronic deposition of monosodium urate (MSU) crystals in the joints and other tissues, resulting in the production of excess reactive oxygen species (ROS) and ...proinflammatory cytokines that intensify synovial inflammation. This condition is mainly associated with inflammatory M1 macrophage activation and oxidative stress production. Hence, gout symptoms can often be resolved by eliminating M1 macrophage activation and scavenging oxidative stress in the inflamed areas. Herein, we developed M1-macrophage-targeting biomineralized metallic nanozymes (FALNZs) that deplete oxidative stress and reduce the M1 macrophage levels to mitigate gouty arthritis. Intra-articular injection of the FALNZs targets inflammatory macrophages and suppresses ROS levels in joints with MSU-crystal-induced arthritis. In addition, the FALNZs alleviate joint swelling, inflammatory cytokine production, and pathological features of the joints. Overall, the proposed therapeutic approach is biocompatible and is an effective ROS scavenger for the treatment of gouty pathogenesis.
Targeted delivery of ceria nanozymes depletes oxidative stress in inflammatory macrophage cells and alleviates gouty arthritis symptoms by suppressing inflammatory signals.
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