HIV-1 Infection Reduces NAD Capping of Host Cell snRNA and snoRNA.

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g., U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity.

LACTB exerts tumor suppressor properties in epithelial ovarian cancer through regulation of Slug.

Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.

LACTB induces cancer cell death through the activation of the intrinsic caspase-independent pathway in breast cancer.

BACKGROUND: LACTB was recently identified as a mitochondrial tumour suppressor that negatively affects cancer cell proliferation by inducing cell death and/or differentiation, depending on the cell type and tissue. However, the detailed mechanism underlying the LACTB-induced cancer cell death is largely unknown. METHODS: We used cell-based, either in 2D or 3D conditions, and in vivo experiments to understand the LACTB mechanisms. In this regard, protein array followed by an enrichment analysis, cell proliferation assays using different compounds, western blot analysis, flow cytometry and immunofluorescence were performed. Differences between quantitative variables following normal distribution were valuated using Student t test for paired or no-paired samples according to the experiment. For in vivo experiments differences in tumour growth were analyzed by 2-way ANOVA. RESULTS: We show, that LACTB expression leads to cell cycle arrest in G1 phase and increase of DNA oxidation that leads to activation of intrinsic caspase-independent cell death pathway. This is achieved by an increase of mitochondrial reactive oxygen species since early time points of LACTB induction. CONCLUSION: Our work provides a deeper mechanistic insight into LACTB-mediated cancer-cell death and shows the dynamics of the cellular responses a particular tumor suppressive stimulus might evoke under different genetic landscapes.

Glutamine synthetase licenses APC/C-mediated mitotic progression to drive cell growth.

Tumors can reprogram the functions of metabolic enzymes to fuel malignant growth; however, beyond their conventional functions, key metabolic enzymes have not been found to directly govern cell mitosis. Here, we report that glutamine synthetase (GS) promotes cell proliferation by licensing mitotic progression independently of its metabolic function. GS depletion, but not impairment of its enzymatic activity, results in mitotic arrest and multinucleation across multiple lung and liver cancer cell lines, patient-derived organoids and xenografted tumors. Mechanistically, GS directly interacts with the nuclear pore protein NUP88 to prevent its binding to CDC20. Such interaction licenses activation of the CDC20-mediated anaphase-promoting complex or cyclosome to ensure proper metaphase-to-anaphase transition. In addition, GS is overexpressed in human non-small cell lung cancer and its depletion reduces tumor growth in mice and increases the efficacy of microtubule-targeted chemotherapy. Our findings highlight a moonlighting function of GS in governing mitosis and illustrate how an essential metabolic enzyme promotes cell proliferation and tumor development, beyond its main metabolic function.

Mitochondrial Tumor Suppressors-The Energetic Enemies of Tumor Progression.

Tumor suppressors represent a critical line of defense against tumorigenesis. Their mechanisms of action and the pathways they are involved in provide important insights into cancer progression, vulnerabilities, and treatment options. Although nuclear and cytosolic tumor suppressors have been extensively investigated, relatively little is known about tumor suppressors localized within the mitochondria. However, recent research has begun to uncover the roles of these important proteins in suppressing tumorigenesis. Here, we review this newly developing field and summarize available information on mitochondrial tumor suppressors.

Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas.

The epithelial-to-mesenchymal transition (EMT) is a cell biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is unclear, however, whether the activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1, and contained within their stroma CD8+ T cells and M1 (antitumor) macrophages. In contrast, tumors arising from more mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1, and contained within their stroma regulatory T cells, M2 (protumor) macrophages, and exhausted CD8+ T cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Finally, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens. Cancer Res; 77(15); 3982-9. ©2017 AACR.

Integrin-ß4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells.

Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-ß4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.

LACTB is a tumour suppressor that modulates lipid metabolism and cell state.

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.

Inflammation Triggers Zeb1-Dependent Escape from Tumor Latency.

The emergence of metastatic disease in cancer patients many years or decades after initial successful treatment of primary tumors is well documented but poorly understood at the molecular level. Recent studies have begun exploring the cell-intrinsic programs, causing disseminated tumor cells to enter latency and the cellular signals in the surrounding nonpermissive tissue microenvironment that maintain the latent state. However, relatively little is known about the mechanisms that enable disseminated tumor cells to escape cancer dormancy or tumor latency. We describe here an in vivo model of solitary metastatic latency in the lung parenchyma. The induction of a localized inflammation in the lungs, initiated by lipopolysaccharide treatment, triggers the awakening of these cells, which develop into macroscopic metastases. The escape from latency is dependent on the expression of Zeb1, a key regulator of the epithelial-to-mesenchymal transition (EMT). Furthermore, activation of the EMT program on its own, as orchestrated by Zeb1, is sufficient to incite metastatic outgrowth by causing carcinoma cells to enter stably into a metastasis-initiating cell state. Cancer Res; 76(23); 6778-84. ©2016 AACR.

Slug and Sox9 cooperatively determine the mammary stem cell state.

Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

AACR special conference on epithelial-mesenchymal transition and cancer progression and treatment.

Epithelial-mesenchymal transition (EMT) is a developmental program implicated in cancer progression and was the subject of the 2010 AACR meeting on the topic of EMT and Cancer Progression and Treatment held on February 28 to March 2 in Arlington, Virginia. A review of the involvement of EMT in gastrulation, organogenesis, carcinogenesis, and metastatic progression elucidated the overlap of EMT in these physiologic and pathologic conditions. Both novel and traditional markers of cells undergoing EMT were discussed and compared with features used to define cancer stem cells. Importantly, these defining characteristics of cells undergoing EMT were discussed in the context of therapeutic and prognostic developments.

Isolation of an active Lv1 gene from cattle indicates that tripartite motif protein-mediated innate immunity to retroviral infection is widespread among mammals.

Lv1/TRIM5alpha (tripartite motif 5alpha) has recently emerged as an important factor influencing species-specific permissivity to retroviral infection in a range of primates, including humans. Old World monkey TRIM5alpha blocks human immunodeficiency virus type 1 (HIV-1) infectivity, and the human and New World monkey TRIM5alpha proteins are inactive against HIV-1 but active against divergent murine (N-tropic murine leukemia virus [MLV-N]) and simian (simian immunodeficiency virus from rhesus macaque [SIVmac]) retroviruses, respectively. Here we demonstrate antiviral activity of the first nonprimate TRIM protein, from cattle, active against divergent retroviruses, including HIV-1. The number of closely related human TRIM sequences makes assignment of the bovine sequence as a TRIM5alpha ortholog uncertain, and we therefore refer to it as bovine Lv1. Bovine Lv1 is closely related to primate TRIM5alpha proteins in the N-terminal RING and B-box 2 domains but significantly less homologous in the C-terminal B30.2 domain, particularly in the region shown to influence antiviral specificity. Intriguingly, some viruses restricted by bovine Lv1, including HIV-1 and MLV-N, are unable to synthesize viral DNA by reverse transcription, whereas restricted HIV-2 makes normal amounts of DNA. The data support the conclusion that TRIM protein-mediated restriction of retroviral infection is a more common attribute of mammals than previously appreciated.

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity.

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.

Retroviral restriction factors Fv1 and TRIM5alpha act independently and can compete for incoming virus before reverse transcription.

The restriction factors Fv1 and TRIM5alpha provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5alpha-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5alpha for virus before reverse transcription. In human cells expressing Fv1(b), N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5alpha by Fv1. Our findings suggest that, like TRIM5alpha, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5alpha when expressed in human cells.

Differential restriction of human immunodeficiency virus type 2 and simian immunodeficiency virus SIVmac by TRIM5alpha alleles.

Primate lentiviruses have narrow host ranges, due in part to their sensitivities to mammalian intracellular antiviral factors such as APOBEC3G and TRIM5alpha. Despite the protection provided by this innate immune system, retroviruses are able to transfer between species where they can cause disease. This is true for sooty mangabey simian immunodeficiency virus, which has transferred to humans as HIV-2 and to rhesus macaques as SIVmac, where it causes AIDS. Here we examine the sensitivities of the closely related HIV-2 and SIVmac to restriction by TRIM5alpha. We show that rhesus TRIM5alpha can restrict HIV-2 but not the closely related SIVmac. SIVmac has not completely escaped TRIM5alpha, as shown by its sensitivity to distantly related TRIM5alpha from the New World squirrel monkey. Squirrel monkey TRIM5alpha blocks SIVmac infection after DNA synthesis and is not saturable with restriction-sensitive virus-like particles. We map the determinant for TRIM5alpha sensitivity to the structure in the capsid protein that recruits CypA into HIV-1 virions. We also make an SIV, mutated at this site, which bypasses restriction in all cells tested.

The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities.

The rhesus macaque tripartite motif containing protein TRIM5alpha specifically restricts HIV-1 infection at an early post-entry step before reverse transcription [Stremlau, M., Owens, C. M., Perron, M. J., Kiessling, M., Autissier, P. & Sodroski, J. (2004) Nature 427, 848-853]. Here, we show that the human and African green monkey (AGM) TRIM5alpha genes encode Ref1 and Lv1 antiretroviral activities, respectively. Expression of TRIM5alpha in permissive cat cells renders them resistant to restriction-sensitive murine leukemia virus but not closely related insensitive virus. Disruption of TRIM5alpha expression in human and AGM cells with small interfering RNA rescues infectivity of restricted virus without affecting unrestricted virus. We also demonstrate that the activity of the murine restriction factor Fv1 depends on TRIM5alpha expression when Fv1 is expressed in human cells. Furthermore, a drug that modifies the behavior of the related promyelocytic leukemia protein PML specifically rescues infection by viruses restricted by human TRIM5alpha. Alignment of the TRIM5alpha proteins from rhesus macaque and AGM indicates an 18-aa insertion. We speculate that this insertion may contribute to the broader specificity of the AGM TRIM5alpha restriction as compared with the human and rhesus macaque proteins.