The androgen deprivation therapy (ADT) to systematically suppress/reduce androgens binding to the androgen receptor (AR) has been the standard therapy for prostate cancer (PCa); yet, most of ADT eventually fails leading to the recurrence of castration resistant PCa. Here, we found that the PCa patients who received ADT had increased PCa stem/progenitor cell population. The addition of the anti-androgen, Casodex®, or AR-siRNA in various PCa cells led to increased stem/progenitor cells, whereas, in contrast, the addition of functional AR led to decreased stem/progenitor cell population but increased non-stem/progenitor cell population, suggesting that AR functions differentially in PCa stem/progenitor vs. non-stem/progenitor cells. Therefore, the current ADT might result in an undesired expansion of PCa stem/progenitor cell population, which explains why this therapy fails. Using various human PCa cell lines and three different mouse models, we concluded that targeting PCa non-stem/progenitor cells with AR degradation enhancer ASC-J9® and targeting PCa stem/progenitor cells with 5-azathioprine and γ-tocotrienol resulted in a significant suppression of the tumors at the castration resistant stage. This suggests that a combinational therapy that simultaneously targets both stem/progenitor and non-stem/progenitor cells will lead to better therapeutic efficacy and may become a new therapy to battle the PCa before and after castration resistant stages.

Dysregulation of microRNAs is a common feature in human cancers, including breast cancer (BC). Here we describe the epigenetic regulation of miR-148a and miR-152 and their impact on BC cells. Due to the hypermethylation of CpG island, the expression levels of both miR-148a and miR-152 (miR-148a/152) are decreased in BC tissues and cells. DNMT1, the DNA methyltransferase 1 for the maintenance methylation, is aberrantly up-regulated in BC and its overexpression is responsible for hypermethylation of miR-148a and miR-152 promoters. Intriguingly, we found that DNMT1 expression, which is one of the targets of miR-148a/152, is inversely correlated with the expression levels of miR-148a/152 in BC tissues. Those results lead us to propose a negative feedback regulatory loop between miR-148a/152 and DNMT1 in BC. More importantly, we demonstrate that IGF-IR and IRS1, often overexpressed in BC, are two novel targets of miR-148a/152. Overexpression of miR-148a or miR-152 significantly inhibits BC cell proliferation, colony formation, and tumor angiogenesis via targeting IGF-IR and IRS1 and suppressing their downstream AKT and MAPK/ERK signaling pathways. Our results suggest a novel miR-148a/152-DNMT1 regulatory circuit and reveal that miR-148a and miR-152 act as tumor suppressors by targeting IGF-IR and IRS1, and that restoration of miR-148a/152 expression may provide a strategy for therapeutic application to treat BC patients.

Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation. A chondrogenic phase is typically observed in this process, which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle. Two prospective cell populations were isolated from rat skeletal muscle: fascia-derived cells (FDCs), extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body. Both populations were investigated for their cell surface marker profiles (flowcytometry analysis), proliferation rates as well as their myogenic and chondrogenic potentials. The majority of FDCs expressed mesenchymal stromal cell markers but not endothelial cell markers. FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro, but not myogenic differentiation. Although MDCs showed chondrogenic potential, they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however, the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium. To clarify the role of the muscle-derived myogenic cells in chondrogenesis, mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential. Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis. Taken together, our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

Osteoclasts (OCs) are responsible for bone resorption in inflammatory joint diseases. Monocyte chemotactic protein-1 (MCP-1) has been shown to induce differentiation of monocytes to OC precursors, but nothing is known about the underlying mechanisms. Here, we elucidate how MCPIP, induced by MCP-1, mediates this differentiation. Knockdown of MCPIP abolished MCP-1-mediated expression of OC markers, tartrate-resistant acid phosphatase, and serine protease cathepsin K. Expression of MCPIP induced p47PHOX and its membrane translocation, reactive oxygen species formation, and induction of endoplasmic reticulum (ER) stress chaperones, up-regulation of autophagy marker, Beclin-1, and lipidation of LC3, and induction of OC markers. Inhibition of oxidative stress attenuated ER stress and autophagy, and suppressed expression of OC markers. Inhibition of ER stress by a specific inhibitor or by knockdown of IRE1 blocked autophagy and induction of OC markers. ER stress inducers, tunicamycin and thapsigargin, induced expression of OC markers. Autophagy inhibition by 3′-methyladenine, LY294002, wortmannin or by knockdown of Beclin-1 or Atg 7 inhibited MCPIP-induced expression of OC markers. These results strongly suggest that MCP-1-induced differentiation of OC precursor cells is mediated via MCPIP-induced oxidative stress that causes ER stress leading to autophagy, revealing a novel mechanistic insight into the role of MCP-1 in OCs differentiation.

Interplay between Foxp3+ regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4+Foxp3+ Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4+Foxp3+ cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in ‘infectious tolerance’ and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.

The field of regenerative medicine is rapidly gaining momentum as an increasing number of reports emerge concerning the induced conversions observed in cellular fate reprogramming. While in recent years, much attention has been focused on the conversion of fate-committed somatic cells to an embryonic-like or pluripotent state, there are still many limitations associated with the applications of induced pluripotent stem cell reprogramming, including relatively low reprogramming efficiency, the times required for the reprogramming event to take place, the epigenetic instability, and the tumorigenicity associated with the pluripotent state. On the other hand, lineage reprogramming involves the conversion from one mature cell type to another without undergoing conversion to an unstable intermediate. It provides an alternative approach in regenerative medicine that has a relatively lower risk of tumorigenesis and increased efficiency within specific cellular contexts. While lineage reprogramming provides exciting potential, there is still much to be assessed before this technology is ready to be applied in a clinical setting.

The DNA damage response (DDR) is a signal transduction pathway that decides the cell's fate either to repair DNA damage or to undergo apoptosis if there is too much damage. Post-translational modifications modulate the assembly and activity of protein complexes during the DDR pathways. MicroRNAs (miRNAs) are emerging as a class of endogenous gene modulators that control protein levels, thereby adding a new layer of regulation to the DDR. In this review, we describe a new role for miRNAs in regulating the cellular response to DNA damage with a focus on DNA double-strand break damage. We also discuss the implications of miRNA's role in the DDR to stem cells, including embryonic stem cells and cancer stem cells, stressing the potential applications for miRNAs to be used as sensitizers for cancer radiotherapy and chemotherapy.

Although cancer and neurodegenerative disease are two distinct pathological disorders, emerging evidence indicates that these two types of disease share common mechanisms of genetic and molecular abnormalities. Recent studies show that individual microRNAs (miRNAs) could be involved in the pathology of both diseases, indicating that the mechanisms of these two seemingly dichotomous diseases converge in the dysregulation of gene expression at the post-transcriptional level. Given the increasing evidence showing that miRNA-based therapeutic strategies that modulate the activity of one or more miRNAs are potentially effective for a wide range of pathological conditions, the involvement of miRNAs in the common pathways of leading both diseases suggests a bright future for developing common therapeutic approaches for both diseases. Moreover, the miRNAs that are dysregulated in both diseases may hold promise as uniquely informative diagnostic markers. Here, we review recent studies on the miRNAs that have been implicated in both cancer and neurodegenerative diseases.

Nuclear factor κB (NF-κB) is a transcriptional factor that regulates a battery of genes that are critical to innate and adaptive immunity, cell proliferation, inflammation, and tumor development. MicroRNAs (miRNAs) are short RNA molecules of 20–25 nucleotides in length that negatively regulate gene expression in animals and plants primarily by targeting 3′ untranslated regions of mRNAs. In this work, we review the convergence of miRNAs and NF-κB signaling and dysregulation of miRNAs and NF-κB activation in human diseases, particularly in cancer. The function of miR-146, miR-155, miR-181b, miR-21, and miR-301a in NF-κB activation and their impact on tumorigenesis are discussed. Given that over 1000 human miRNAs have been identified, rendering miRNAs one of the most abundant classes of regulatory molecules, deciphering their biological function and pathological contribution in NF-κB dysregulation is essential to appreciate the complexity of immune systems and to develop therapeutics against cancer.

Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.

Tumor suppressor p53 plays a central role in tumor prevention. As a transcription factor, p53 mainly exerts its function through transcription regulation of its target genes to initiate various cellular responses. To maintain its proper function, p53 is tightly regulated by a wide variety of regulators in cells. Thus, p53, its regulators and regulated genes form a complex p53 network which is composed of hundreds of genes and their products. microRNAs (miRNAs) are a class of endogenously expressed, small non-coding RNA molecules which play a key role in regulation of gene expression at the post-transcriptional level. Recent studies have demonstrated that miRNAs interact with p53 and its network at multiple levels. p53 regulates the transcription expression and the maturation of a group of miRNAs. On the other hand, miRNAs can regulate the activity and function of p53 through direct repression of p53 or its regulators in cells. These findings have demonstrated that miRNAs are important components in the p53 network, and also added another layer of complexity to the p53 network.

High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks. Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their roles in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku, DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its role in fork restart. Most cancer therapeutic strategies create DNA replication stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.

DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints. Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability.

‘Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

BRCA1 plays a critical role in the regulation of homologous recombination (HR)-mediated DNA double-strand break repair. BRCA1-deficient cancers have evolved to tolerate loss of BRCA1 function. This renders them vulnerable to agents, such as PARP inhibitors, that are conditionally ‘synthetic lethal' with their underlying repair defect. Recent studies demonstrate that BRCA1-deficient cells may acquire resistance to these agents by partially correcting their defect in HR-mediated repair, either through reversion mutations in BRCA1 or through ‘synthetic viable' loss of 53BP1. These findings and their clinical implications will be reviewed in this article.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing β-cells in the pancreatic islets. There is an immediate need to restore both β-cell function and immune tolerance to control disease progression and ultimately cure T1D. Currently, there is no effective treatment strategy to restore glucose regulation in patients with T1D. FoxP3-expressing CD4+ regulatory T cells (Tregs) are potential candidates to control autoimmunity because they play a central role in maintaining self-tolerance. However, deficiencies in either naturally occurring Tregs (nTregs) themselves and/or their ability to control pathogenic effector T cells have been associated with T1D. Here, we hypothesize that nTregs can be replaced by FoxP3+ adaptive Tregs (aTregs), which are uniquely equipped to combat autoreactivity in T1D. Unlike nTregs, aTregs are stable and provide long-lived protection. In this review, we summarize the current understanding of aTregs and their potential for use as an immunological intervention to treat T1D.

T lymphocytes bearing the αβ T cell receptor (TCR) but lacking CD4, CD8, and markers of natural killer (NK) cell differentiation are known as ‘double-negative’ (DN) T cells and have been described in both humans and rodent models. We and others have shown that DN T cells can act as regulatory T cells (Tregs) that are able to prevent allograft rejection, graft-versus-host disease, and autoimmune diabetes. In the last few years, new data have revealed evidence of DN Treg function in vivo in rodents and humans. Moreover, significant advances have been made in the mechanisms by which DN Tregs target antigen-specific T cells. One major limitation of the field is the lack of a specific marker that can be used to distinguish truly regulatory DN T cells (DN Tregs) from non-regulatory ones, and this is the central challenge in the coming years. Here, we review recent progress on the role of DN Tregs in transplantation and autoimmunity, and their mechanisms of action. We also provide some perspectives on how DN Tregs compare with Foxp3+ Tregs.

Foxp3+ T regulatory cells (Tregs) consisting of natural and induced Treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The actions designed to correct defects in numbers or functions of Tregs may be therapeutic in the treatment of autoimmune diseases. While recent studies demonstrated that natural Tregs are instable and dysfunctional in the inflammatory condition, induced Tregs (iTregs) may have a different feature. Here we review the progress of iTregs, particularly focus on their stability and function in the established autoimmune diseases. The advantage of iTregs as therapeutics used under inflammatory conditions is highlighted. Proper generation and manipulation of iTregs used for cellular therapy may provide a promise for the treatment of many autoimmune and inflammatory diseases.

Regulators of G-protein signaling (RGS) proteins are scaffolds that control diverse signaling pathways by modulating signalosome formation and by accelerating the GTPase activity of heterotrimeric G proteins. Although expression of many RGS proteins is relatively low in quiescent cells, transcriptional and post-translational responses to environmental cues regulate both their abundance and activity. We found previously that RGS13, one of the smallest RGS proteins in the family, inhibited cyclic AMP-dependent protein kinase (PKA)-induced gene expression through interactions with the transcription factor cAMP-response element-binding (CREB) protein. Here, we show that PKA activation also leads to increased steady-state RGS13 expression through RGS13 phosphorylation, which inhibits RGS13 protein degradation. RGS13 turnover was significantly reduced in cells stimulated with cAMP, which was reversed by expression of the PKA-specific inhibitory peptide PKI. RGS13 phosphorylation was diminished by mutation of an N-terminal Thr residue (T41) identified as a phosphorylation site by mass spectrometry. Mutation of Thr41 in RGS13 to Ala (T41A) reduced steady-state RGS13 levels and its ability to inhibit M2 muscarinic receptor-mediated Erk phosphorylation compared with wild-type RGS13 by attenuating the protective effect of cAMP on RGS13 degradation. RGS13 underwent ubiquitylation, indicating that it is a likely target of the proteasome. These studies are the first to demonstrate post-translational mechanisms controlling the expression of RGS13. Stabilization of RGS13 through PKA-mediated phosphorylation could enhance RGS13 functions, providing negative feedback regulation that promotes cellular desensitization.

During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. Accurate attachment of spindle microtubules to kinetochore requires the chromosomal passenger of Aurora B kinase complex with borealin, INCENP and survivin (SUR). The current working model argues that SUR is responsible for docking Aurora B to the centromere whereas its precise role in Aurora B activation has been unclear. Here, we show that Aurora B kinase activation requires SUR priming phosphorylation at Ser20 which is catalyzed by polo-like kinase 1 (PLK1). Inhibition of PLK1 kinase activity or expression of non-phosphorylatable SUR mutant prevents Aurora B activation and correct spindle microtubule attachment. The PLK1-mediated regulation of Aurora B kinase activity was examined in real-time mitosis using fluorescence resonance energy transfer-based reporter and quantitative analysis of native Aurora B substrate phosphorylation. We reason that the PLK1-mediated priming phosphorylation is critical for orchestrating Aurora B activity in centromere which is essential for accurate chromosome segregation and faithful completion of cytokinesis.

Autophagy down-regulates the Wnt signal transduction pathway via targeted degradation of a key signaling protein. This may provide an explanation for autophagy's role in tumor suppression.

Completion of lagging strand DNA synthesis requires processing of up to 50 million Okazaki fragments per cell cycle in mammalian cells. Even in yeast, the Okazaki fragment maturation happens approximately a million times during a single round of DNA replication. Therefore, efficient processing of Okazaki fragments is vital for DNA replication and cell proliferation. During this process, primase-synthesized RNA/DNA primers are removed, and Okazaki fragments are joined into an intact lagging strand DNA. The processing of RNA/DNA primers requires a group of structure-specific nucleases typified by flap endonuclease 1 (FEN1). Here, we summarize the distinct roles of these nucleases in different pathways for removal of RNA/DNA primers. Recent findings reveal that Okazaki fragment maturation is highly coordinated. The dynamic interactions of polymerase δ, FEN1 and DNA ligase I with proliferating cell nuclear antigen allow these enzymes to act sequentially during Okazaki fragment maturation. Such protein–protein interactions may be regulated by post-translational modifications. We also discuss studies using mutant mouse models that suggest two distinct cancer etiological mechanisms arising from defects in different steps of Okazaki fragment maturation. Mutations that affect the efficiency of RNA primer removal may result in accumulation of unligated nicks and DNA double-strand breaks. These DNA strand breaks can cause varying forms of chromosome aberrations, contributing to development of cancer that associates with aneuploidy and gross chromosomal rearrangement. On the other hand, mutations that impair editing out of polymerase α incorporation errors result in cancer displaying a strong mutator phenotype.

RAD9 regulates multiple cellular processes that influence genomic integrity, and for at least some of its functions the protein acts as part of a heterotrimeric complex bound to HUS1 and RAD1 proteins. RAD9 participates in DNA repair, including base excision repair, homologous recombination repair and mismatch repair, multiple cell cycle phase checkpoints and apoptosis. In addition, functions including the transactivation of downstream target genes, immunoglobulin class switch recombination, as well as 3′–5′ exonuclease activity have been reported. Aberrant RAD9 expression has been linked to breast, lung, thyroid, skin and prostate tumorigenesis, and a cause–effect relationship has been demonstrated for the latter two. Interestingly, human RAD9 overproduction correlates with prostate cancer whereas deletion of Mrad9, the corresponding mouse gene, in keratinocytes leads to skin cancer. These results reveal that RAD9 protein can function as an oncogene or tumor suppressor, and aberrantly high or low levels can have deleterious health consequences. It is not clear which of the many functions of RAD9 is critical for carcinogenesis, but several alternatives are considered herein and implications for the development of novel cancer therapies based on these findings are examined.