Potential application of cell reprogramming techniques for cancer research.

The ability to control the transition from an undifferentiated stem cell to a specific cell fate is one of the key techniques that are required for the application of interventional technologies to regenerative medicine and the treatment of tumors and metastases and of neurodegenerative diseases. Reprogramming technologies, which include somatic cell nuclear transfer, induced pluripotent stem cells, and the direct reprogramming of specific cell lineages, have the potential to alter cell plasticity in translational medicine for cancer treatment. The characterization of cancer stem cells (CSCs), the identification of oncogene and tumor suppressor genes for CSCs, and the epigenetic study of CSCs and their microenvironments are important topics. This review summarizes the application of cell reprogramming technologies to cancer modeling and treatment and discusses possible obstacles, such as genetic and epigenetic alterations in cancer cells, as well as the strategies that can be used to overcome these obstacles to cancer research.

Positive Feedback Loop of OCT4 and c-JUN Expedites Cancer Stemness in Liver Cancer.

The network of stemness genes and oncogenes in human patient-specific reprogrammed cancer stem cells (CSCs) remains elusive, especially in liver cancer. HepG2-derived induced pluripotent stem cell-like cells (HepG2-iPS-like cells) were generated by introducing Yamanaka factors and the knockdown vector shTP53. They exhibited features of stemness and a higher tumorigenesis after xenograft transplantation compared with HepG2 cells. The cancerous mass of severe combined immunodeficiency (SCID) mice derived from one colony was dissected and cultured to establish reprogrammed HepG2-derived CSC-like cells (designated rG2-DC-1C). A single colony exhibited 42% occurrence of tumors with higher proliferation capacities. rG2-DC-1C showed continuous expression of the OCT4 stemness gene and of representative tumor markers, potentiated chemoresistance characteristics, and invasion activities. The sphere-colony formation ability and the invasion activity of rG2-DC-1C were also higher than those of HepG2 cells. Moreover, the expression of the OCT4 gene and the c-JUN oncogene, but not of c-MYC, was significantly elevated in rG2-DC-1C, whereas no c-JUN expression was observed in HepG2 cells. The positive-feedback regulation via OCT4-mediated transactivation of the c-JUN promoter and the c-JUN-mediated transactivation of the OCT4 promoter were crucial for promoting cancer development and maintaining cancer stemness in rG2-DC-1C. Increased expression of OCT4 and c-JUN was detected in the early stage of human liver cancer. Therefore, the positive feedback regulation of OCT4 and c-JUN, resulting in the continuous expression of oncogenes such as c-JUN, seems to play a critical role in the determination of the cell fate decision from iPS cells to CSCs in liver cancer. Stem Cells 2016;34:2613-2624.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Syndromic features and mild cognitive impairment in mice with genetic reduction on p300 activity: Differential contribution of p300 and CBP to Rubinstein-Taybi syndrome etiology.

Rubinstein-Taybi syndrome (RSTS) is a complex autosomal-dominant disease characterized by mental and growth retardation and skeletal abnormalities. A majority of the individuals diagnosed with RSTS carry heterozygous mutation in the gene CREBBP, but a small percentage of cases are caused by mutations in EP300. To investigate the contribution of p300 to RSTS pathoetiology, we carried out a comprehensive and multidisciplinary characterization of p300(+/-) mice. These mice exhibited facial abnormalities and impaired growth, two traits associated to RSTS in humans. We also observed abnormal gait, reduced swimming speed, enhanced anxiety in the elevated plus maze, and mild cognitive impairment during the transfer task in the water maze. These analyses demonstrate that p300(+/-) mice exhibit phenotypes that are reminiscent of neurological traits observed in RSTS patients, but their comparison with previous studies on CBP deficient strains also indicates that, in agreement with the most recent findings in human patients, the activity of p300 in cognition is likely less relevant or more susceptible to compensation than the activity of CBP.

Jdp2-deficient granule cell progenitors in the cerebellum are resistant to ROS-mediated apoptosis through xCT/Slc7a11 activation.

The Jun dimerization protein 2 (Jdp2) is expressed predominantly in granule cell progenitors (GCPs) in the cerebellum, as was shown in Jdp2-promoter-Cre transgenic mice. Cerebellum of Jdp2-knockout (KO) mice contains lower number of Atoh-1 positive GCPs than WT. Primary cultures of GCPs from Jdp2-KO mice at postnatal day 5 were more resistant to apoptosis than GCPs from wild-type mice. In Jdp2-KO GCPs, the levels of both the glutamate‒cystine exchanger Sc7a11 and glutathione were increased; by contrast, the activity of reactive oxygen species (ROS) was decreased; these changes confer resistance to ROS-mediated apoptosis. In the absence of Jdp2, a complex of the cyclin-dependent kinase inhibitor 1 (p21Cip1) and Nrf2 bound to antioxidant response elements of the Slc7a11 promoter and provide redox control to block ROS-mediated apoptosis. These findings suggest that an interplay between Jdp2, Nrf2, and p21Cip1 regulates the GCP apoptosis, which is one of critical events for normal development of the cerebellum.

Author Correction: Jdp2-deficient granule cell progenitors in the cerebellum are resistant to ROS-mediated apoptosis through xCT/Slc7a11 activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cdk1-mediated phosphorylation of human ATF7 at Thr-51 and Thr-53 promotes cell-cycle progression into M phase.

Activating transcription factor 2 (ATF2) and its homolog ATF7 are phosphorylated at Thr-69/Thr-71 and at Thr-51/Thr-53, respectively, by stress-activated MAPKs regulating their transcriptional functions in G1 and S phases. However, little is known about the role of ATF2 and ATF7 in G2/M phase. Here, we show that Cdk1-cyclin B1 phosphorylates ATF2 at Thr-69/Thr-71 and ATF7 at Thr-51/Thr-53 from early prophase to anaphase in the absence of any stress stimulation. Knockdown of ATF2 or ATF7 decreases the rate of cell proliferation and the number of cells in M-phase. In particular, the knockdown of ATF7 severely inhibits cell proliferation and G2/M progression. The inducible expression of a mitotically nonphosphorylatable version of ATF7 inhibits G2/M progression despite the presence of endogenous ATF7. We also show that mitotic phosphorylation of ATF7 promotes the activation of Aurora kinases, which are key enzymes for early mitotic events. These results suggest that the Cdk1-mediated phosphorylation of ATF7 facilitates G2/M progression, at least in part, by enabling Aurora signaling.

Control of Oxidative Stress and Generation of Induced Pluripotent Stem Cell-like Cells by Jun Dimerization Protein 2.

We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knock-out mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.

GCN5 and p300 share essential functions during early embryogenesis.

Previous studies revealed that deletion of genes encoding the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality in mice. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of histone acetyltransferases interact functionally in vivo, we created mice lacking one or more alleles of p300, GCN5, or PCAF. As expected, we found that mice heterozygous for any single null allele are viable. The majority of GCN5(+/-)p300(+/-) mice also survive to adulthood with no apparent abnormalities. However, approximately 25% of these mice die before birth. These embryos are developmentally stunted and exhibit increased apoptosis compared with wild-type or single GCN5(+/-) or p300(+/-) littermates at embryonic day 8.5. In contrast, no abnormalities were observed in PCAF(-/-) p300(+/-) mice. Of interest, we find that p300 protein levels vary in different mouse genetic backgrounds, which likely contributes to the incomplete penetrance of the abnormal phenotype of GCN5(+/-) p300(+/-) mice. Our data indicate that p300 cooperates specifically with GCN5 to provide essential functions during early embryogenesis.

Essential function of p300 acetyltransferase activity in heart, lung and small intestine formation.

p300 and CBP are large nuclear acetyltransferases exhibiting a complex multi-domain structure. Mouse embryos nullizygous for either p300 or Cbp die at midgestation, while heterozygotes are viable but in part display defects in neurulation or bone morphogenesis. To directly examine the contribution of the acetyltransferase (AT) activity to mouse development, we have abrogated this function by a knock-in approach. Remarkably, a single AT-deficient allele of p300 or Cbp leads to embryonic or neonatal lethality, indicating that the mutant alleles are dominant. Formation of the cardiovascular system, the lung and the small intestine are strongly impaired in p300 AT and to a much lesser extent in Cbp AT mutant embryos, a difference that is also reflected by the defects in gene expression. Embryonic stem cells homozygous for either the p300 AT or a p300 null mutation respond differently to BMP2 stimulation, indicating that the two alleles are not equivalent. Unexpectedly, the p300 AT-mutant cells upregulate BMP-inducible genes to levels similar or even higher than observed in wild-type cells.

Differential role of p300 and CBP acetyltransferase during myogenesis: p300 acts upstream of MyoD and Myf5.

Studies in tissue culture cells have implicated p300 and CBP acetyltransferases in myogenic regulatory factor (MRF) mediated transcription and terminal differentiation of skeletal muscle cells. However, in vivo data placing p300 and CBP on myogenic differentiation pathways are not yet available. In this report we provide genetic evidence that p300 but not CBP acetyltransferase (AT) activity is required for myogenesis in the mouse and in embryonic stem (ES) cells. A fraction of embryos carrying a single p300 AT- deficient allele exhibit impaired MRF expression, delayed terminal differentiation and a reduced muscle mass. In mouse embryos lacking p300 protein, Myf-5 induction is severely attenuated. Similarly, ES cells homozygous for a p300 AT or a p300 null mutation fail to activate Myf5 and MyoD transcription efficiently, while Pax3, acting genetically upstream of these MRFs, is expressed. In contrast, ES cells lacking CBP AT activity express MyoD and Myf5 and undergo myogenic differentiation. These data reveal a specific requirement for p300 and its AT activity in the induction of MRF gene expression and myogenic cell fate determination in vivo.