Characterization Of Induced Neural Progenitors From Skin Fibroblasts By A Novel Combination Of Defined Factors

Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders

Characterization of induced neural progenitors from skin fibroblasts by a novel combination of defined factors.

Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy-resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer

Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies

In vivo modeling of metastatic human high-grade serous ovarian cancer in mice

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies

Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

<div><p>DNA methyltransferase 3A (DNMT3A) is an enzyme involved in DNA methylation that is frequently mutated in human hematologic malignancies. We have previously shown that inactivation of Dnmt3a in hematopoietic cells results in chronic lymphocytic leukemia in mice. Here we show that 12% of Dnmt3a-deficient mice develop CD8+ mature peripheral T cell lymphomas (PTCL) and 29% of mice are affected by both diseases. 10% of <i>Dnmt3a</i>^<i>+/-</i> mice develop lymphomas, suggesting that Dnmt3a is a haploinsufficient tumor suppressor in PTCL. DNA methylation was deregulated genome-wide with 10-fold more hypo- than hypermethylated promoters and enhancers, demonstrating that hypomethylation is a major event in the development of PTCL. Hypomethylated promoters were enriched for binding sites of transcription factors AML1, NF-κB and OCT1, implying the transcription factors potential involvement in Dnmt3a-associated methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting that cancer-specific hypomethylation has broader effects on the transcriptome of cancer cells than hypermethylation. Interestingly, transcriptomes of <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old <i>Dnmt3a</i>^<i>+/-</i> tumor-free mice, suggesting that p53 downregulation is chronologically an intermediate event in tumorigenesis. Decrease in p53 is likely an important event in tumorigenesis because its overexpression inhibited proliferation in mouse PTCL cell lines, suggesting that low levels of p53 are important for tumor maintenance. Altogether, our data link the haploinsufficient tumor suppressor function of Dnmt3a in the prevention of mouse mature CD8+ PTCL indirectly to a <i>bona fide</i> tumor suppressor of T cell malignancies p53.</p></div

Promoter and gene-body hypomethylation is present throughout the genome of <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCLs.

<p>Circos plot of DMRS annotated to promoters and gene bodies in <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL relative to <i>wild-type</i> CD8+ cells. DMRs aligning to promoter (outer circle) and gene body (inner circles) are displayed in relation to its chromosomal position in the mouse genome. Hypomethylated DMRS are indicated by yellow lines and hypermethylated DMRS are indicated by blue lines.</p

Jdp2 is hypomethylated and overexpressed in human and mouse PTCL.

<p>(A) COBRA analysis of mouse Jdp2 promoter methylation in three independent <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown. (B) Normalized gene expression of Jdp2 in mouse CD8+ control, <i>Dnmt3a</i>^<i>+/-</i> PTCL, and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test). (C) Bisulfite sequencing of the JDP2 promoter in normal human CD3+ T cells and in two independent human PTCL samples. Circles represent individual CpGs within the promoter. Black and white areas denote the relative portion of methylated and un-methylated sequence reads at a CpG, respectively. (D) Normalized gene expression of JDP2 in normal human CD3+ T cells and human PTCL samples, by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test).</p