A Targeted Deep Sequencing Method to Quantify Endogenous Retrovirus Gag Sequence Variants and Open Reading Frames Expressed in Nonobese Diabetic Mice.

Endogenous retroviruses (ERVs) are involved in autoimmune diseases such as type 1 diabetes (T1D). ERV gene products homologous to murine leukemia retroviruses are expressed in the pancreatic islets of NOD mice, a model of T1D. One ERV gene, Gag, with partial or complete open reading frames (ORFs), is detected in the islets, and it contains many sequence variants. An amplicon deep sequencing analysis was established by targeting a conserved region within the Gag gene to compare NOD with T1D-resistant mice or different ages of prediabetic NOD mice. We observed that the numbers of different Gag variants and ORFs are linked to T1D susceptibility. More importantly, these numbers change during the course of diabetes development and can be quantified to calculate the levels of disease progression. Sequence alignment analysis led to identification of additional markers, including nucleotide mismatching and amino acid consensus at specific positions that can distinguish the early and late stages, before diabetes onset. Therefore, the expression of sequence variants and ORFs of ERV genes, particularly Gag, can be quantified as biomarkers to estimate T1D susceptibility and disease progression.

Endogenous retrovirus Gag antigen and its gene variants are unique autoantigens expressed in the pancreatic islets of non-obese diabetic mice.

Endogenous retrovirus (ERV) are remnants of ancient retroviruses that have been incorporated into the genome and evidence suggests that they may play a role in the etiology of T1D. We previously identified a murine leukemia retrovirus-like ERV whose Env and Gag antigens are involved in autoimmune responses in non-obese diabetic (NOD) mice. In this study, we show that the Gag antigen is present in the islet stromal cells. Although Gag gene transcripts were present, Gag protein was not detected in diabetes-resistant mice. Cloning and sequencing analysis of individual Gag genes revealed that NOD islets express Gag gene variants with complete open-reading frames (ORFs), in contrast to the diabetes-resistant mice, whose islet Gag gene transcripts are mostly non-ORFs. Importantly, the ORFs obtained from the NOD islets are extremely heterogenous, coding for various mutants that are absence in the genome. We further show that Gag antigens are stimulatory for autoreactive T cells and identified one islet-expressing Gag variant that contains an altered peptide ligand capable of inducing IFN-gamma release by the T cells. The data highlight a unique retrovirus-like factor in the islets of the NOD mouse strain, which may participate in key events triggering autoimmunity and T1D.

miR-30 disrupts senescence and promotes cancer by targeting both p16INK4A and DNA damage pathways.

miR-30 is a microRNA frequently overexpressed in human cancers. However, the biological consequence of miR-30 overexpression in cancer has been unclear. In a genetic screen, miR-30 was found to abrogate oncogenic-induced senescence, a key tumor-suppressing mechanism that involves DNA damage responses, activation of p53 and induction of p16INK4A. In cells and mouse models, miR-30 disrupts senescence and promotes cancer by suppressing 2 targets, CHD7 and TNRC6A. We show that while CHD7 is a transcriptional coactivator essential for induction of p16INK4A in senescent cells, TNRC6A, a miRNA machinery component, is required for expression and functionality of DNA damage response RNAs (DDRNAs) that mediate DNA damage responses and p53 activation by orchestrating histone modifications, chromatin remodeling and recruitment of DNA damage factors at damaged sites. Thus, miR-30 inhibits both p16INK4A and p53, 2 key senescence effectors, leading to efficient senescence disruption. These findings have identified novel signaling pathways mediating oncogene-induced senescence and tumor-suppression, and revealed the molecular and cellular mechanisms underlying the oncogenic activity of miR-30. Thus, the miR-30/CHD7/TNRC6A pathway is potentially a novel diagnostic biomarker and therapeutic target for cancer.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice.

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis.

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.

The epithelial-mesenchymal transition mediator S100A4 maintains cancer-initiating cells in head and neck cancers.

Cancer-initiating cells (CIC) comprise a rare subpopulation of cells in tumors that are proposed to be responsible for tumor growth. Starting from CICs identified in head and neck squamous cell carcinomas (HNSCC), termed head and neck cancer-initiating cells (HN-CIC), we determined as a candidate stemness-maintaining molecule for HN-CICs the proinflammatory mediator S100A4, which is also known to be an inducer of epithelial-mesenchymal transition. S100A4 knockdown in HN-CICs reduced their self-renewal capability and their stemness and tumorigenic properties, both in vitro and in vivo. Conversely, S100A4 overexpression in HNSCC cells enhanced their stem cell properties. Mechanistic investigations indicated that attenuation of endogenous S100A4 levels in HNSCC cells caused downregulation of Notch2 and PI3K (phosphoinositide 3-kinase)/pAKT along with upregulation of PTEN, consistent with biological findings. Immunohistochemical analysis of HNSCC clinical specimens showed that S100A4 expression was positively correlated with clinical grading, stemness markers, and poorer patient survival. Together, our findings reveal a crucial role for S100A4 signaling pathways in maintaining the stemness properties and tumorigenicity of HN-CICs. Furthermore, our findings suggest that targeting S100A4 signaling may offer a new targeted strategy for HNSCC treatment by eliminating HN-CICs.

The miR-17-92 cluster of microRNAs confers tumorigenicity by inhibiting oncogene-induced senescence.

In mammalian cells, activation of oncogenes usually triggers innate tumor-suppressing defense mechanisms, including apoptosis and senescence, which are compromised by additional mutations before cancers are developed. The miR-17-92 gene cluster, a polycistron encoding six microRNAs (miRNA), is frequently overexpressed in human cancers and has been shown to promote several aspects of oncogenic transformation, including evasion of apoptosis. In the current study, we show a new role of miR-17-92 in inhibiting oncogenic ras-induced senescence. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for this antisenescence activity. miR-17 and miR-20a are both necessary and sufficient for conferring resistance to ras-induced senescence by directly targeting p21(WAF1), a key effector of senescence. By contrast, these components are not essential for the ability of miR-17-92 to evade Myc-induced apoptosis. Moreover, disruption of senescence by miR-17-92 or its miR-17/20a components leads to enhanced oncogenic transformation by activated ras in primary human cells. Taken together with previous reports that miR-17-92 inhibits apoptosis by suppressing Pten via the miR-19 components, our results indicate that this miRNA cluster promotes tumorigenesis by antagonizing both tumor-suppressing mechanisms, apoptosis, and senescence, through the activities of different miRNA components encoded in this cluster.

Tid1 functions as a tumour suppressor in head and neck squamous cell carcinoma.

Human tumourous imaginal disc (Tid1), a human homologue of the Drosophila tumour suppressor protein Tid56, is involved in multiple intracellular signalling pathways such as apoptosis, cell proliferation, and cell survival. Here, we investigated the anti-tumourigenic activity of Tid1 in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Firstly, the clinical association between Tid1 expression and progression of HNSCC was explored. It was found that expression of Tid1 was negatively associated with tumour status, recurrence, and survival prognosis using immunohistochemical analysis of primary HNSCC patient tumour tissue. Secondly, ectopic expression of Tid1 in HNSCC cells was shown to significantly inhibit cell proliferation, migration, invasion, anchorage-independent growth, and xenotransplantation tumourigenicity. Thirdly, we showed that overexpression of Tid1 attenuated EGFR activity and blocked the activation of AKT in HNSCC cells, which are known to be involved in the regulation of survival in HNSCC cells. On the other hand, ectopic expression of constitutively active AKT greatly reduced apoptosis induced by Tid1 overexpression. Together, these findings suggest that Tid1 functions as a tumour suppressor in HNSCC tumourigenesis.

RNA interference against retroviruses.

Bang and Ellerman, and later Peyton Rous, reported the first identification of transmissible cancer-causing agents, which later turned out to be avian retroviruses. Today avian retroviruses are important models for study of retrovirus replication and pathogenesis, and also important pathogens of domestic fowl. Here we describe the use of RNA interference (RNAi) in live chick embryos to block replication of an avian retrovirus. We also describe inhibition of ASLV and HIV replication in cell culture with RNAi.

Inhibition of retroviral pathogenesis by RNA interference.

BACKGROUND: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates. RESULTS: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive. CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.