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The MYC oncogene is often dysregulated in human cancer, including hepatocellular carcinoma (HCC). MYC is considered undruggable to date. Here, we comprehensively identify genes essential for survival of MYChigh but not MYClow cells by a CRISPR/Cas9 genome-wide screen in a MYC-conditional HCC model. Our screen uncovers novel MYC synthetic lethal (MYC-SL) interactions and identifies most MYC-SL genes described previously. In particular, the screen reveals nucleocytoplasmic transport to be a MYC-SL interaction. We show that the majority of MYC-SL nucleocytoplasmic transport genes are upregulated in MYChigh murine HCC and are associated with poor survival in HCC patients. Inhibiting Exportin-1 (XPO1) in vivo induces marked tumor regression in an autochthonous MYC-transgenic HCC model and inhibits tumor growth in HCC patient-derived xenografts. XPO1 expression is associated with poor prognosis only in HCC patients with high MYC activity. We infer that MYC may generally regulate and require altered expression of nucleocytoplasmic transport genes for tumorigenesis.

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The MYC proto-oncogenes encode a family of transcription factors that are among the most commonly activated oncoproteins in human neoplasias. Indeed, MYC aberrations or upregulation of MYC-related pathways by alternate mechanisms occur in the vast majority of cancers. MYC proteins are master regulators of cellular programmes. Thus, cancers with MYC activation elicit many of the hallmarks of cancer required for autonomous neoplastic growth. In preclinical models, MYC inactivation can result in sustained tumour regression, a phenomenon that has been attributed to oncogene addiction. Many therapeutic agents that directly target MYC are under development; however, to date, their clinical efficacy remains to be demonstrated. In the past few years, studies have demonstrated that MYC signalling can enable tumour cells to dysregulate their microenvironment and evade the host immune response. Herein, we discuss how MYC pathways not only dictate cancer cell pathophysiology but also suppress the host immune response against that cancer. We also propose that therapies targeting the MYC pathway will be key to reversing cancerous growth and restoring antitumour immune responses in patients with MYC-driven cancers.

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The tetracycline regulatory system provides a tractable strategy to interrogate the role of oncogenes in the initiation, maintenance, and regression of tumors through both spatial and temporal control of expression. This approach has several potential advantages over conventional methods to generate genetically engineered mouse models. First, continuous constitutive overexpression of an oncogene can be lethal to the host impeding further study. Second, constitutive overexpression fails to model adult onset of disease. Third, constitutive deletion does not permit, whereas conditional overexpression of an oncogene enables the study of the consequences of restoring expression of an oncogene back to endogenous levels. Fourth, the conditional activation of oncogenes enables examination of specific and/or developmental state-specific consequences.Hence, by allowing precise control of when and where a gene is expressed, the tetracycline regulatory system provides an ideal approach for the study of putative oncogenes in the initiation as well as the maintenance of tumorigenesis and the examination of the mechanisms of oncogene addiction. In this protocol, we describe the methods involved in the development of a conditional mouse model of MYC-induced T-cell acute lymphoblastic leukemia.

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Myc and Ras are two of the most commonly activated oncogenes in tumorigenesis. Together and independently they regulate many cancer hallmarks including proliferation, apoptosis, and self-renewal. Recently, they were shown to cooperate to regulate host tumor microenvironment programs including host immune responses. But, is their partnership always cooperative or do they have distinguishable functions? Here, we provide one perspective that Myc and Ras cooperation depends on the genetic evolution of a particular cancer. This in turn, dictates when they cooperate via overlapping and identifiably distinct cellular- and host immune-dependent mechanisms that are cancer type specific.

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Inhibition of cancer cell adhesion is an effective approach to killing adherent cancer cells. B49 and its analog B49Mod1 peptides, derived from the extracellular domain (ECD) of bone marrow stromal antigen 2 (BST-2), display anti-adhesion activity on breast cancer cells. However, the minimal sequence required for this anti-adhesion activity is unknown. Here, we further characterized the anti-adhesion activity of B49Mod1. We show that the anti-adhesion activity of B49Mod1 may require cysteine-linked disulfide bond and that the peptide is susceptible to proteolytic deactivation. Using structure-activity relationship studies, we identified an 18-Mer sequence (B18) as the minimal peptide sequence mediating the anti-adhesion activity of B49Mod1. Atomistic molecular dynamic (MD) simulations reveal that B18 forms a stable complex with the ECD of BST-2 in aqueous solution. MD simulations further reveal that B18 may cause membrane defects that facilitates peptide translocation across the bilayer. Placement of four B18 chains as a transmembrane bundle results in water channel formation, indicating that B18 may impair membrane integrity and form pores. We hereby identify B18 as the minimal peptide sequence required for the anti-adhesion activity of B49Mod1 and provide atomistic insight into the interaction of B18 with BST-2 and the cell membrane.

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Bone marrow stromal antigen 2 (BST-2) mediates various facets of cancer progression and metastasis. Here, we show that BST-2 is linked to poor survival in invasive breast cancer patients as its expression positively correlates with disease severity. However, the mechanisms that drive the pro-metastatic functions of BST-2 are not fully understood. Correlation of BST-2 expression and tumor aggressiveness was analyzed in human tissue samples. Migration, invasion, and competitive experimental metastasis assays were used to measure the cellular responses after silencing BST-2 expression. Using a mouse model of breast cancer, we show that BST-2 promotes metastasis independent of the primary tumor. Additional experiments show that suppression of BST-2 renders non-adherent cancer cells non-viable by sensitizing cells to anoikis. Embedment of cancer cells in basement membrane matrix reveals that silencing BTS-2 expression inhibits invadopodia formation, extracellular matrix degradation, and subsequent cell invasion. Competitive experimental pulmonary metastasis shows that silencing BST-2 reduces the numbers of viable circulating tumor cells (CTCs) and decreases the efficiency of lung colonization. Our data define a previously unknown function for BST-2 in the i) formation of invadopodia, ii) degradation of extracellular matrix, and iii) protection of CTCs from hemodynamic stress. We believe that physical (tractional forces) and biochemical (ECM type/composition) cues may control BST-2's role in cell survival and invadopodia formation. Collectively, our findings highlight BST-2 as a key factor that allows cancer cells to invade, survive in circulation, and at the metastatic site.

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Bone marrow stromal antigen 2 (BST-2) also known as Tetherin has been implicated in the growth and progression of many cancers. BST-2 employs its pro-tumor effects through the formation of BST-2:BST-2 dimers which ultimately promotes cell to cell and cell to matrix adhesion, cell motility, survival, and growth. The aim of this study was to evaluate the effect of a novel BST-2-based peptide-B49 on adhesion and growth of breast cancer cells. Homotypic/heterotypic adhesion, three-dimensional spheroid formation, and anchorage-independent growth were used to assess the effect of B49 on cell adhesion and growth. Additionally, we provide evidence of the anti-tumor effect of B49 in a preclinical mouse model of breast cancer. Results show that breast cancer cell adhesion to other cancer cells or components of the tumor microenvironment were inhibited by B49. Most well-known evaluation indexes of cancer cell growth, including spheroid formation, anchorage-independent, and primary tumor growth were significantly inhibited by B49. These data affirm that i) BST-2 plays a key role in mediating breast cancer cell adhesion and growth, and ii) B49 and its analog B49Mod1 significantly inhibits BST-2-mediated cancer cell adhesion and growth. Therefore, B49 and its analogs offer a promising anti-adhesion and therapeutic lead for BST-2-dependent cancers.

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Almost all breast tumors express the antiviral protein BST-2 with 67%, 25% and 8.2% containing high, medium or low levels of BST-2, respectively. Breast tumor cells and tissues that contain elevated levels of BST-2 are highly aggressive. Suppression of BST-2 expression reprograms tumorigenic properties of cancer cells and diminishes cancer cell aggressiveness. Using structure/function studies, we report that dimerization of BST-2 through cysteine residues located in the BST-2 extracellular domain (ECD), leads to anoikis resistance and cell survival through proteasome-mediated degradation of BIM-a key proapoptotic factor. Importantly, BST-2 dimerization promotes tumor growth in preclinical breast cancer models in vitro and in vivo. Furthermore, we demonstrate that restoration of the ECD cysteine residues is sufficient to rescue cell survival and tumor growth via a previously unreported pathway-BST-2/GRB2/ERK/BIM/Cas3. These findings suggest that disruption of BST-2 dimerization offers a potential therapeutic approach for breast cancer.

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There is now irrefutable evidence that overexpression of the innate immunity protein-BST-2, in breast cancer cells is implicated in tumor growth and progression. The cellular mechanisms that control BST-2-mediated effect in tumor progression involve enhancement of cancer cell motility-migration/invasion. However, the distinct structural elements of BST-2 that mediate breast cancer cell motility remain unknown. Here, we used various motility assays and different variants of BST-2 to examine the cellular and structural mechanisms controlling BST-2-mediated cell motility. We show that BST-2 silencing in various cancer cell lines inhibits cell motility. Restoration of BST-2 expression using construct expressing wild type BST-2 rescues cell motility. Mutational analysis identifies the cytoplasmic tail of BST-2 as a novel regulator of cancer cell motility, because cell motility was significantly abrogated by substitution of the BST-2 cytoplasmic tail tyrosine residues to alanine residues. Furthermore, in a spheroid invasion model, BST-2-expressing tumor spheroids are highly invasive inside 3D Matrigel matrices. In this model, the spreading distance of BST-2-expressing spheroids was significantly higher than that of BST-2-suppressed spheroids. Collectively, our data reveal that i) BST-2-expressing breast cancer cells in spheroids are more motile than their BST-2-supressed counterparts; ii) BST-2 cytoplasmic tail regulates non-proteolytic (migration) and proteolytic (invasion) mechanisms of breast cancer cell motility; and iii) replacement of the tyrosine residues at positions 6 and 8 in the cytoplasmic tail of BST-2 with alanine residues inhibits cell motility.

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Host cells respond to viral infections by activating immune response genes that are not only involved in inflammation, but may also predispose cells to cancerous transformation. One such gene is BST-2, a type II transmembrane protein with a unique topology that endows it tethering and signaling potential. Through this ability to tether and signal, BST-2 regulates host response to viral infection either by inhibiting release of nascent viral particles or in some models inhibiting viral dissemination. However, despite its antiviral functions, BST-2 is involved in disease manifestation, a function linked to the ability of BST-2 to promote cell-to-cell interaction. Therefore, modulating BST-2 expression and/or activity has the potential to influence course of disease.

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BACKGROUND: Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells. MATERIALS AND METHODS: We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines. RESULTS: We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA. CONCLUSION: Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer.

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INTRODUCTION: Several innate immunity genes are overexpressed in human cancers and their roles remain controversial. Bone marrow stromal antigen 2 (BST-2) is one such gene whose role in cancer is not clear. BST-2 is a unique innate immunity gene with both antiviral and pro-tumor functions and therefore can serve as a paradigm for understanding the roles of other innate immunity genes in cancers. METHODS: Meta-analysis of tumors from breast cancer patients obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets were evaluated for levels of BST-2 expression and for tumor aggressiveness. In vivo, we examined the effect of knockdown of BST-2 in two different murine carcinoma cells on tumor growth, metastasis, and survival. In vitro, we assessed the effect of carcinoma cell BST-2 knockdown and/or overexpression on adhesion, anchorage-independent growth, migration, and invasion. RESULTS: BST-2 in breast tumors and mammary cancer cells is a strong predictor of tumor size, tumor aggressiveness, and host survival. In humans, BST-2 mRNA is elevated in metastatic and invasive breast tumors. In mice, orthotopic implantation of mammary tumor cells lacking BST-2 increased tumor latency, decreased primary tumor growth, reduced metastases to distal organs, and prolonged host survival. Furthermore, we found that the cellular basis for the role of BST-2 in promoting tumorigenesis include BST-2-directed enhancement in cancer cell adhesion, anchorage-independency, migration, and invasion. CONCLUSIONS: BST-2 contributes to the emergence of neoplasia and malignant progression of breast cancer. Thus, BST-2 may (1) serve as a biomarker for aggressive breast cancers, and (2) be a novel target for breast cancer therapeutics.

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Bone marrow stromal antigen 2 (BST-2; also known as tetherin or CD317) is an IFN-inducible gene that functions to block the release of a range of nascent enveloped virions from infected host cells. However, the role of BST-2 in viral pathogenesis remains poorly understood. BST-2 plays a multifaceted role in innate immunity, as it hinders retroviral infection and possibly promotes infection with some rhabdo- and orthomyxoviruses. This paradoxical role has probably hindered exploration of BST-2 antiviral function in vivo. We reported previously that BST-2 tethers Chikungunya virus (CHIKV)-like particles on the cell plasma membrane. To explore the role of BST-2 in CHIKV replication and host protection, we utilized CHIKV strain 181/25 to examine early events during CHIKV infection in a BST-2(-/-) mouse model. We observed an interesting dichotomy between WT and BST-2(-/-) mice. BST-2 deficiency increased inoculation site viral load, culminating in higher systemic viraemia and increased lymphoid tissues tropism. A suppressed inflammatory innate response demonstrated by impaired expression of IFN-α, IFN-γ and CD40 ligand was observed in BST-2(-/-) mice compared with the WT controls. These findings suggested that, in part, BST-2 protects lymphoid tissues from CHIKV infection and regulates CHIKV-induced inflammatory response by the host.

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BST-2 restricts MMTV replication, but once infection has established, MMTV modulates BST-2 levels. MMTV-directed BST-2 modulation is tissue-specific and dependent on infection and neoplastic transformation status of cells. In the lymphoid compartment of infected mice, BST-2 expression is first upregulated and then significantly downregulated regardless of absence or presence of mammary tumors. However, in mammary gland tissues, upregulation of BST-2 expression is dependent on the presence of mammary tumors and tumor tissues themselves have high BST-2 levels. Elevated BST-2 expression in these tissues is not attributable to IFN since levels of IFNα and IFNγ negatively correlate with BST-2. Importantly, soluble factors released by tumor cells suppress IFNα and IFNγ but induce BST-2. These data suggest that overexpression of BST-2 in carcinoma tissues could not be attributed to IFNs but to a yet to be determined factor that upregulates BST-2 once oncogenesis is initiated.

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