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Introduction: Fusobacterium necrophorum, a human and animal pathogen, is the primary etiologic agent of bovine liver abscesses and a driving factor for prophylactic antibiotic use in the fed cattle industry. Considering calls to reduce agricultural antibiotic use, we isolated phages capable of killing F. necrophorum as an alternative or complementary biocontrol strategy. Methods: Six novel phages (φFN37, φRTG5, φKSUM, φHugo, φPaco, and φBB) were isolated from rumen fluid or ruminal F. necrophorum isolates and subjected to host range testing on both F. necrophorum subspecies. Four F. necrophorum subspecies, necrophorum phages, were tested for cross-resistance and host growth inhibition individually and in pairs. Additionally, genomic sequencing, annotation, and analysis were performed.s. Results: Four of six isolated phages were able to form lysogens, although all six contained lysogeny-related genes. φKSUM and φBB, did not form lysogens and were able to infect both subspecies. Four phages could infect F. necrophorum 8L1 (a liver abscess model challenge strain) in vitro. Genomic analysis showed that these phages belong to class Caudoviricetes with genome sizes ranging from 35 kbp to 111 kbp and GC values ranging from 26% to 36% and have extremely limited similarity to other deposited phage genomes infecting Fusobacterium or other genera. Conclusions: Although all phages isolated contained sequences bearing similarities to genes implicated in lysogeny, the four selected for use in cocktails showed potential in inhibiting host growth, with several demonstrating promising attributes for biocontrol and therapeutic applications. Phage cocktails that may offer enhanced antibacterial activity were also identified, indicating the potential of some lysogenic phages to be adapted for biocontrol or therapeutic purposes when lytic phages are difficult to obtain.
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Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A by Zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages towards an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that Zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon response uniformly across models. The induction of an interferon response is partially due to the inhibition of Sox4 translation by Zotatifin. A similar induction of interferon-stimulated genes was observed in breast cancer patient biopsies following Zotatifin treatment. Surprisingly, Zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened interferon response resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for Zotatifin, and provide a rationale for new combination regimens comprising Zotatifin and chemotherapy or immunotherapy as treatments for TNBC.
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Protein synthesis is frequently dysregulated in cancer and selective inhibition of mRNA translation represents an attractive cancer therapy. Here, we show that therapeutically targeting the RNA helicase eIF4A with zotatifin, the first-in-class eIF4A inhibitor, exerts pleiotropic effects on both tumor cells and the tumor immune microenvironment in a diverse cohort of syngeneic triple-negative breast cancer (TNBC) mouse models. Zotatifin not only suppresses tumor cell proliferation but also directly repolarizes macrophages toward an M1-like phenotype and inhibits neutrophil infiltration, which sensitizes tumors to immune checkpoint blockade. Mechanistic studies revealed that zotatifin reprograms the tumor translational landscape, inhibits the translation of Sox4 and Fgfr1, and induces an interferon (IFN) response uniformly across models. The induction of an IFN response is partially due to the inhibition of Sox4 translation by zotatifin. A similar induction of IFN-stimulated genes was observed in breast cancer patient biopsies following zotatifin treatment. Surprisingly, zotatifin significantly synergizes with carboplatin to trigger DNA damage and an even heightened IFN response, resulting in T cell-dependent tumor suppression. These studies identified a vulnerability of eIF4A in TNBC, potential pharmacodynamic biomarkers for zotatifin, and provide a rationale for new combination regimens consisting of zotatifin and chemotherapy or immunotherapy as treatments for TNBC.
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Antineoplásicos , Neoplasias de Mama Triplo Negativas , Animais , Camundongos , Humanos , Interferons , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Proliferação de Células , Microambiente TumoralRESUMO
Tumor dormancy is a stage in which residual cancer cells remain inactive, but regrowth of dormant cancer cells contributes to recurrence. The complex ecosystem in cancer that promotes cell survival and the factors that eventually overcome growth constraints and result in proliferation remain to be fully elucidated. Doing so may provide new insights and help identify novel strategies to prolong cancer dormancy and prevent disease recurrence. To dissect the molecular pathways and the microenvironments involved in regulation of dormancy, we utilized a novel immunocompetent transgenic model to study minimal residual disease and relapse. This model revealed a significant reorganization of cancer cell structures, stroma, and immune cells, with cancer cells showing dormant cell signatures. Single-cell RNA sequencing uncovered remodeling of myeloid and lymphoid compartments. In addition, the Jagged-1/Notch signaling pathway was shown to regulate many aspects of tumorigenesis, including stem cell development, epithelial-to-mesenchymal transition, and immune cell homeostasis during minimal residual disease. Treatment with an anti-Jagged-1 antibody inhibited the Jagged-1/Notch signaling pathway in tumor cells and the microenvironment, delaying tumor recurrence. These findings uncover a cascade of regulatory changes in the microenvironment during dormancy and identify a therapeutic strategy to undercut these changes. SIGNIFICANCE: Single-cell RNA-sequencing analysis reveals dormancy-associated changes in immune and stromal cells and demonstrates a rationale to pursue Jagged-1/Notch pathway inhibition as a viable therapeutic strategy to reduce disease recurrence.
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Ecossistema , Análise de Célula Única , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/genética , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE: Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models.
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Neoplasias de Mama Triplo Negativas , Animais , Linfócitos B , Claudinas/metabolismo , Claudinas/uso terapêutico , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Humanos , Macrófagos/metabolismo , Camundongos , Linfócitos T Citotóxicos/patologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents embryo implantation and leads to infertility. Analysis of Acvr2a-PRcre and Acvr2b-PRcre pregnant mice determined that BMP signaling occurs via ACVR2A and that ACVR2B is dispensable during embryo implantation. Therefore, BMPs signal through a conserved endometrial ACVR2A/SMAD1/5 pathway that promotes endometrial receptivity during embryo implantation.
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Proteínas Morfogenéticas Ósseas/metabolismo , Implantação do Embrião , Infertilidade Feminina/genética , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animais , Biópsia , Modelos Animais de Doenças , Endométrio/metabolismo , Endométrio/patologia , Estrogênios/metabolismo , Feminino , Humanos , Camundongos , Camundongos Knockout , Gravidez , Transdução de Sinais/fisiologia , Proteína Smad1/análise , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/análise , Proteína Smad5/genética , Proteína Smad5/metabolismoRESUMO
Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, frequent loss of chromosome 5q11-35 which includes PLK2 is observed in basal-like breast cancer. In this study, we found that PLK2 was tumor suppressive in breast cancer, preferentially in basal-like and triple-negative breast cancer (TNBC) subtypes. Knockdown of PLK1 rescued phenotypes induced by PLK2-loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase through the kinase but not the polo-box domains of PLK2, suggesting PLK2 functioned at least partially through the interaction with PLK1. Furthermore, an improved treatment response was seen in both Plk2-deleted/low mouse preclinical and PDX TNBC models using the PLK1 inhibitor volasertib alone or in combination with carboplatin. Re-expression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. In summary, this study delineates the effects of chromosome 5q loss in TNBC that includes PLK2, the relationship between PLK2 and PLK1, and how this may render PLK2-deleted/low tumors more sensitive to PLK1 inhibition in combination with chemotherapy.