Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.