Spindle assembly checkpoint and p53 deficiencies cooperate for tumorigenesis in mice

The spindle assembly checkpoint (SAC) guards against chromosomal missegregation during mitosis. To investigate the role of SAC in tumor development, mice heterozygously knocked out for the mitotic arrest deficient (Mad) genes Mad1 and/or Mad2 were mated with p53^+/? mice. Increased tumor frequencies were reproducibly observed in Mad2^+/?p53^+/? (88.2%) and Mad1^+/?Mad2^+/?p53^+/? (95.0%) mice compared with p53^+/? (66.7%) mice. Moreover, 53% of Mad2^+/?p53^+/? mice developed lymphomas compared with 11% of p53^+/? mice. By examining chromosome content, increased loss in diploidy was seen in cells from Mad2^+/?p53^+/? versus p53^+/? mice, correlating loss of SAC function, in a p53^+/? context, with increased aneuploidy and tumorigenesis. The findings here provide evidence for a cooperative role of Mad1/Mad2 and p53 genes in preventing tumor development. Published 2008 Wiley‐Liss, Inc.     

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice

Introduction

Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm ^+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm ^+/- mice on the p53 ^+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation.

Methods

BALB/cHeA-p53 ^+/- mice were crossed with MSM/Ms-Atm ^+/- mice, and females of the F₁ progeny ([BALB/cHeA × MSM/Ms]F₁) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks.

Results

We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53 ^+/- mice of the BALB/cHeA × MSM/Ms background. The singly heterozygous p53 ^+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53 ^+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma.

Conclusion

Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53 ^+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.     

The tissue dependent interactions between p53 and Bcl-2 in vivo

To further investigate the role of p53 in apoptosis in vivo and the interaction between p53 and Bcl-2 in the regulation of cellular apoptosis in vivo, we depleted p53 in Bcl-2-null mice. We found that the interaction between p53 and Bcl-2 are tissue dependent. Specifically, loss of p53 in Bcl-2^−/− mice inhibits apoptotic induction in spleen and subsequently inhibits the Bcl-2-null-induced spleen atrophy. Furthermore, p53 deficiency overcomes loss of melanocyte stem cell (MSC)-induced apoptosis and subsequently prevents hair graying in Bcl-2- null mice. In addition, p53 deletion partly inhibits apoptosis in hair follicle keratinocytes, leading to the alleviation of hair growth delay in Bcl-2-null mice. However, p53 absence in Bcl-2-null mice cannot restore other defects in Bcl-2-null mice, including retardation of growth, short ears and polycystic kidney disease.     

Tumor suppressor genes promote rhabdomyosarcoma progression in p53 heterozygous,HER-2/neu transgenic mice

Human sarcomas arise suddenly, thus preempting the study of preneoplastic and early neoplastic lesions. To explore the natural history of these tumors we studied male mice carrying a heterozygous deletion of p53 and an activated HER-2/neu transgene (BALB-p53Neu mice), that develop urethral rhabdomyosarcomas with nearly full penetrance and early onset (4 months of age). Among genes prominently upregulated in preneoplastic tissue, and more highly expressed in tumors, we found the insulin-like growth factor 2 (Igf2) and tumor suppressors, p19Arf and p21Cip1. In urethral tissues of male mice p53 was less expressed than in female mice, whereas HER-2/neu was more expressed, a combination not found in other skeletal muscles of the same mice that could contribute to the anatomic and sexual specificity of BALB-p53Neu rhabdomyosarcoma. Upregulation of p19Arf and p21Cip1 was additively determined by HER-2/neu activation and by p53 inactivation. Silencing of p19Arf or p21Cip1 in rhabdomyosarcoma cell lines can inhibit cell growth and motility, thus suggesting that these genes can contribute to growth autonomy and malignancy of tumor cells. In vivo injection of gene-silenced cells highlighted selective variations in organ-specific metastatic ability, indicating that overexpression of p19Arf and p21Cip1 controlled both tumor cell-intrinsic properties and microenvironmental interactions. The onset of pelvic rhabdomyosarcoma in BALB-p53Neu male mice is triggered by the coincidental overexpression of HER-2/neu and hypoexpression of the residual p53 allele, that foster p53 loss, Igf2 autocriny and overexpression of p19Arf and p21Cip1, a phenotype that could provide novel potential targets for cancer prevention and therapy.     

Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium

Loss of p53 function is an early event in colitis-associatedneoplasia in humans. We assessed the role of p53 in a mousemodel of colitis-associated neoplasia. Colitis was induced inp53^–/–, p53^+/– and p53^+/+ mice using threeor four cycles of dextran sulfate sodium (DSS) followed by 120days of water. Mice were examined for incidence, multiplicityand types of neoplastic lesions. Lesions were examined for mutationsin ß-catenin (exon 3), K-ras (codons 12/13) and p53(exons 5–8) by sequencing and for cellular localizationof ß-catenin by immunohistochemistry. The incidenceof neoplastic lesions was 57, 20 and 20% in p53^–/–,p53^+/– and p53^+/+ mice, respectively (P = 0.013). p53^–/–mice had a greater number of total lesions (P < 0.0001),cancers (P = 0.001) and dysplasias (P = 0.009) per mouse thaneither p53^+/– or p53^+/+ mice. Flat lesions were associatedwith the p53^–/– genotype, whereas polypoid lesionswere associated with the p53^+/– and p53^+/+ genotypes (P< 0.0001). ß-Catenin mutations were present in75% of lesions of p53^+/+ mice and absent in lesions from p53^–/–mice (P = 0.055). Nuclear expression of ß-cateninwas seen only in polypoid lesions (91%). No K-ras or p53 mutationswere detected. These data indicate that loss of p53 enhancesthe induction of colitis-associated neoplasia, particularlyflat lesions, and dysregulation of ß-catenin signalingplays an important role in the formation of polypoid lesionsin this mouse model. As observed in humans, p53 plays a protectiverole in colitis-associated neoplasia in the DSS model. Abbreviations: CRC, colorectal cancer; DSS, dextran sulfate sodium; PCR, polymerase chain reaction; UC, ulcerative colitis; PGE₂, prostaglandin E₂ Received January 24, 2007; revised May 11, 2007; accepted May 29, 2007.     

Shh and p50/Bcl3 signaling crosstalk drives pathogenesis of BCCs in gorlin syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder that is due, in large measure, to aberrant Shh signaling driven by mutations in the tumor suppressor gene Ptch1. Here, we describe the development of Ptch1^+/−/SKH-1 mice as a novel model of this disease. These animals manifest many features of NBCCS, including developmental anomalies and are remarkably sensitive to both ultraviolet (UVB) and ionizing radiation that drive the development of multiple BCCs. Just as in patients with NBCCS, Ptch1^+/−/SKH-1 also spontaneously develops BCCs and other neoplasms such as rhabdomyomas/rhabdomyosarcomas. Administration of smoothened inhibitors (vismodegib/itraconazole/cyclopamine) or non-steroidal anti-inflammatory drug (sulindac/sulfasalazine) each result in partial resolution of BCCs in these animals. However, combined administration of these agents inhibits the growth of UVB-induced BCCs by >90%. Employing small molecule- and decoy-peptide-based approaches we further affirm that complete remission of BCCs could only be achieved by combined inhibition of p50-NFκB/Bcl3 and Shh signaling. We posit that Ptch1^+/−/SKH-1 mice are a novel and relevant animal model for NBCCS. Understanding mechanisms that govern genetic predisposition to BCCs should facilitate our ability to identify and treat NBCCS gene carriers, including those at risk for sporadic BCCs while accelerating development of novel therapeutic modalities for these patients.     

Mammary gland-specific ablation of focal adhesion kinase reduces the incidence of p53-mediated mammary tumour formation

Background:

Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development.

Methods:

To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53^lox/lox/FAK^+/+/WapCre, p53^lox/lox/FAK^flox/+/WapCre and p53^lox/lox/FAK^flox/−/WapCre mice, and mice with WapCre-mediated conditional expression of p53^R270H, the mouse equivalent of human p53^R273H hot spot mutation, together with conditional deletion of FAK, P53^R270H/+/FAK^lox/+/WapCre and p53^R270H/+/FAK^flox/−/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours.

Results:

Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53^R270H-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures.

Conclusions:

Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53^R270H mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53^R270H-induced mammary tumour development.     

Reduced p53 dosage associated with mammary tumor metastases in C3(1)/TAG transgenic mice

Transgenic mice expressing the simian virus 40 large T-antigen (T_AG) under the regulatory control of the rat prostatic steroid binding protein C3(1) gene develop mammary carcinomas (in females) and prostate carcinomas (in males). The development of carcinomas occurs several months after the appearance of dysplastic lesions, suggesting that T_AG is necessary but insufficient for tumor formation and that other genetic events are involved in this process. T_AG is known to bind to p53 and to result in its functional inactivation, which is believed to be a critical step in T_AG-induced transformation. We investigated whether the T_AG-p53 interaction is rate limiting in the development of phenotypic changes in these transgenic mice by crossing C3(1)/T_AG transgenics with mice carrying null mutations of the p53 gene. T_AG-expressing animals with a p53^+/– genotype developed much more aggressive mammary tumors, as evidenced by increased numbers and size of metastases, than did T_AG-expressing animals carrying two wild-type p53 alleles. While p53 was expressed in primary tumors, p53 expression appeared to be reduced or lost in many metastases in mice carrying either the p53^+/+ or p53^+/– genotypes. The tumorigenic process did not appear to be due to the loss of the second wild-type p53 allele or the gain of dominant oncogenic mutations in p53, as no mutations were detected in either primary or metastatic tumors by polymerase chain reaction–single-strand conformation polymorphism analyses. These findings suggest that despite the presence of T_AG, p53 levels influence the characteristics of the late stages of mammary tumor growth and accelerate metastases. Functional loss of p53 and not p53 mutations participates in T_AG-induced mammary carcinoma development and progression. Mol. Carcinog. 20:168–174, 1997. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work, and, as such, is in the public domain in the United States of America.     

Exposure to 60 Hz magnetic fields and risk of lymphoma in PIM transgenic and TSG-p53 (p53 knockout) mice

The results of a number of epidemiology studies suggest that exposure to power frequency (50 and 60 Hz) magnetic fields may be a risk factor for hematopoietic neoplasia. To generate experimental data to test this hypothesis, the influence of magnetic field exposure on lymphoma induction was determined in two strains of mice that are genetically predisposed to the disease. PIM mice, which carry the pim-1 oncogene, are highly sensitive to lymphoma induction by N-ethyl-N-nitrosourea (ENU); ENU-treated PIM mice were studied as a 'high incidence' lymphoma model. TSG-p53 (p53 knockout) mice, in which the p53 tumor suppressor gene has been deleted from the germ line, develop lymphoma as an age- related change; hemizygous TSG-p53 mice were studied as a 'low incidence' lymphoma model. Beginning 1 day after a single i.p. injection of 25 mg ENU/kg body wt, groups of 30 PIM mice/sex were exposed for 18.5 h/day to pure, linearly polarized, transient-free 60 Hz magnetic fields at field strengths of 0 (sham control), 0.02, 2.0 or 10.0 Gauss (G). An additional group of 30 PIM mice/sex was exposed intermittently (1 h on, 1 h off) to 10.0 G fields. Groups of 30 TSG-p53 mice/sex were exposed continuously to magnetic field strengths of 0 (sham control) or 10.0 G; TSG-p53 mice received no ENU. Studies were terminated after 23 weeks of magnetic field exposure. Lymphoma incidence in male PIM mice exposed continuously to 10.0 G magnetic fields was significantly reduced from that seen in sex-matched sham controls; survival, lymphoma incidence and lymphoma latency in other groups of PIM mice did not differ from sham controls. Survival and lymphoma incidence in all groups of TSG-p53 mice was 7% or less, regardless of magnetic field exposure regimen. These data do not support the hypothesis that exposure to magnetic fields is a significant risk factor for lymphoid neoplasia in mice with a genetic predisposition to the disease.      

Tumorigenesis in p27/p53- and p18/p53-double null mice: functional collaboration between the pRb and p53 pathways

Mice lacking both p18(Ink4c) and p27(Kip1) develop a tumor spectrum similar to pRb(+/-) mice, and loss of p53 function accelerates tumorigenesis in pRb(+/-) mice. We hypothesized that codeletion of either p18 or p27 in conjunction with p53 deletion will also accelerate tumorigenesis. Mice lacking both p18 and p53 develop several tumors not reported in either single null genotype, including hepatocellular carcinoma, testicular choriocarcinoma, hemangiosarcoma, leiomyosarcoma, fibrosarcoma, and osteosarcoma. Mice lacking both p27 and p53 exhibit a decreased lifespan and develop unique tumors, including papillary carcinoma of the colon, hemangiosarcoma, and leiomyosarcoma. In both p18/p53 and p27/p53 double null genotypes, the incidence and spectra of tissues that develop lymphoma are also increased, as compared to the single null genotypes. The development of p27/p53 double null colon tumors correlates with secondary changes in cell-cycle protein expression and CDK (cyclin-dependent kinase) activity, perhaps contributing to the progression of colorectal cancer. We concluded that p18 and p27 can, not only functionally collaborate with one another, but also can independently collaborate with p53 to modulate the cell cycle and suppress tumorigenesis in a tissue-specific manner.     

Mice with alterations in both p53 and Ink4a/Arf display a striking increase in lung tumor multiplicity and progression: differential chemopreventive effect of budesonide in wild-type and mutant A/J mice

p53 transgenic mice carrying a dominant negative mutation were crossed with Ink4A/Arf heterozygous-deficient mice to investigate whether there is a synergy between these two germ-line mutations in promoting carcinogen-induced lung tumor progression in mice. Mice with a p53 dominant negative mutation and Ink4A/Arf heterozygous deficiency exhibited >20-fold increase in tumor volume compared with approximately 4-fold increase in Ink4A/Arf heterozygous-deficient mice and a 9-fold increase in mice with only the p53 dominant negative mutation. The effect of Ink4A/Arf heterozygous deficiency on lung tumor progression occurred late in the carcinogenesis process (>30 weeks after carcinogen treatment). In addition, most of the lung tumors (approximately 80%) from mice with a p53 mutation and deletion of Ink4A/Arf were lung adenocarcinomas. In contrast, lung adenocarcinomas were seen in <10% of the lung tumors from the wild-type mice and approximately 50% of the lung tumors from Ink4a/Arf heterozygous-deficient or p53 mutant mice. These results indicate a significant synergistic interaction between the presence of a mutant p53 transgene and the Ink4A/Arf deletion during lung tumor progression (P < 0.01). The usefulness of this new mouse model in lung cancer chemoprevention was examined. The chemopreventive efficacy of budesonide was examined in wild-type mice, mice with Ink4A/Arf heterozygous deficiency, mice with a mutation in the p53 gene, or mice with both a mutation in the p53 gene and deletion in the Ink4A/Arf locus. Mice treated with budesonide displayed an average of 90% inhibition of lung tumor progression in a standard 18-week chemoprevention assay, regardless of p53 and/or Ink4A/Arf status. However, the efficacy of budesonide against lung tumor progression decreased from 94 to 77% (P = 0.07) in mice with alterations in both p53 and Ink4A/Arf in a 40-week chemoprevention assay. Similarly, when mice bearing established lung adenomas were treated with budesonide, genotype-dependent differential effects of budesonide in wild-type and mutant mice were clearly revealed with a 82, 64, 45, and 33% decrease in tumor volume in wild-type mice, p53(+/+)Ink4a/Arf(+/-) mice, p53(+/-)Ink4a/Arf(+/+) mice, and p53(+/-)Ink4a/Arf(+/-), respectively. Thus, mutant mice with alterations in p53 and/or Ink4A/Arf exhibited a significant resistance to chemoprevention by budesonide. Because p53 and Ink4a/Arf mutations are the most prevalent mutations in human lung cancers, the effectiveness of chemopreventive agents on the mutant A/J mice containing alterations with p53 and Ink4a/Arf is the best preclinical estimate of their efficacy in humans. Thus, the mutant A/J mouse model should prove useful for chemoprevention studies.     

Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with Kras^G12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic Kras^G12D expression. Kras^G12D overexpression transformed p53^−/− myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53^−/− myoblasts transformed by Kras^G12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.     

Azathioprine induction of tumors with microsatellite instability: Risk evaluation using a mouse model

Mismatch-repair (MMR)-deficient cells show increased in vitro tolerance to thiopurines because they escape apoptosis resulting from MMR-dependent signaling of drug-induced DNA damage. Prolonged treatment with immunosuppressants including azathioprine (Aza), a thiopurine prodrug, has been suggested as a risk factor for the development of late onset leukemias/lymphomas displaying a microsatellite instability (MSI) phenotype, the hallmark of a defective MMR system. We performed a dose effect study in mice to investigate the development of MSI lymphomas associated with long term Aza treatment. Over two years, Aza was administered to mice that were wild type, null or heterozygous for the MMR gene Msh2. Ciclosporin A, an immunosuppressant with an MMR-independent signaling, was also administered to Msh2^wt mice as controls. Survival, lymphoma incidence and MSI tumor phenotype were investigated. Msh2^+/− mice were found more tolerant than Msh2^wt mice to the cytotoxicity of Aza. In Msh2^+/− mice, Aza induced a high incidence of MSI lymphomas in a dose-dependent manner. In Msh2^wt mice, a substantial lifespan was only observed at the lowest Aza dose. It was associated with the development of lymphomas, one of which displayed the MSI phenotype, unlike the CsA-induced lymphomas. Our findings define Aza as a risk factor for an MSI-driven lymphomagenesis process.     

Alterations of the p53 and pRB pathways in human astrocytoma

Human astrocytomas are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRB and the p53 pathways. Genetic alterations functionally eliminate pRB and p53 themselves or upstream and/or downstream molecules such as products of theInk4a/ARF locus, p16^Ink4a and p14^ARF. As a result, malignant cells are defective in critical cell cycle and apoptosis regulatory elements contributing to unrelenting tumour growth and invasion. Current research aims to discover effective means of reconstituting p53 and pRB pathway components in an effort to attenuate the aggressive phenotype of astrocytoma.     

Loss of p53 Induces Tumorigenesis in p21-Deficient Mesenchymal Stem Cells

There is growing evidence about the role of mesenchymal stem cells (MSCs) as cancer stem cells in many sarcomas. Nevertheless, little is still known about the cellular and molecular mechanisms underlying MSCs transformation. We aimed at investigating the role of p53 and p21, two important regulators of the cell cycle progression and apoptosis normally involved in protection against tumorigenesis. Mesenchymal stem cells from wild-type, p21^-/-p53^+/+, and p21^-/-p53^+/- mice were cultured in vitro and analyzed for the appearance of tumoral transformation properties after low, medium, and high number of passages both in vitro and in vivo. Wild-type or p21^-/-p53^+/+ MSCs did not show any sign of tumoral transformation. Indeed, after short-term in vitro culture, wild-type MSCs became senescent, and p21^-/-p53^+/+ MSCs showed an elevated spontaneous apoptosis rate. Conversely, MSCs carrying a mutation in one allele of the p53 gene (p21^-/-p53^+/- MSCs) completely lost p53 expression after in vitro long-term culture. Loss of p53 was accompanied by a significant increase in the growth rate, gain of karyotypic instability, loss of p16 expression, and lack of senescence response. Finally, these cells were able to form fibrosarcomas partially differentiated into different mesenchymal lineages when injected in immunodeficient mice both after subcutaneous and intrafemoral injection. These findings show that MSCs are very sensitive to mutations in genes involved in cell cycle control and that these deficiencies can be at the origin of some mesodermic tumors.     

Homocysteine induces podocyte apoptosis by regulating miR‐1929‐5p expression through c‐Myc,DNMT1 and EZH2

Chronic kidney disease (CKD) is a common and complex disease in kidneys which has been associated with an increased risk of renal cell carcinoma. Elevated homocysteine (Hcy) levels are known to influence the development and progression of CKD by regulating podocyte injury and apoptosis. To investigate the molecular mechanisms triggered in podocytes by Hcy, we used cbs^+/− mice and observed that higher Hcy levels increased the apoptosis rate of podocytes with accompanying glomerular damage. Hcy‐induced podocyte injury and apoptosis in cbs^+/− mice was regulated by inhibition of microRNA (miR)‐1929‐5p expression. Overexpression of miR‐1929‐5p in podocytes inhibited apoptosis by upregulating Bcl‐2. Furthermore, the expression of miR‐1929‐5p was regulated by epigenetic modifications of its promoter. Hcy upregulated DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) levels, resulting in increased DNA methylation and H3K27me3 levels on the miR‐1929‐5p promoter. Additionally, we observed that c‐Myc recruited DNMT1 and EZH2 to the miR‐1929‐5p promoter and suppressed the expression of miR‐1929‐5p. In summary, we demonstrated that Hcy promotes podocyte apoptosis through the regulation of the epigenetic modifiers DNMT1 and EZH2, which are recruited by c‐Myc to the promoter of miR‐1929‐5p to silence miR‐1929‐5p expression.     

Inflammation regulates microRNA expression in cooperation with p53 and nitric oxide

microRNA (miRNA) are small non‐coding RNA targeting mRNAs leading to their instability and diminished translation. Altered expression of miRNA is associated with cancer. Inflammation and nitric oxide modulates the development of lymphomas in p53 knockout mice and there exists a negative feedback loop between p53 and NOS2. Using a genetic strategy, we tested the hypothesis that inflammation‐induced oxidative and nitrosative stress modulates miRNA expression in mouse model deficient in either p53 or NOS2. Mice treated with Corynebacterium parvum (C. parvum), to induce inflammation, clearly separated from controls by their miRNA profiles in wild‐type, p53‐ and NOS2‐knockout genetic backgrounds. C. parvum‐induced inflammation significantly (p < 0.005) increased miR‐21, miR‐29b and miR‐34a/b/c and decreased (p < 0.005) mir‐29c and mir‐181a/c expression in the spleen of C57BL mice. However, p53‐knockout C57BL mice did not show a significant increase in the mir‐34b/c or a decrease in mir‐29c expression following C. parvum‐induced inflammation. Expression of mir‐21, mir‐29b and mir‐181a was independent of p53‐status. NOS2‐knockout C57BL mice showed a significant increase in miR‐21 and miR‐34a/b/c and decrease in miR‐181a similar to the wild‐type (WT) mice following C. parvum‐induced inflammation. However, in contrast to the WT mice, miR‐29b/c expression was not affected following C. parvum‐induced inflammation in NOS2 knockout mice. N‐acetyl cysteine, an anti‐oxidant, reduced the expression of miR‐21 and miR‐29b in C. parvum‐treated WT mice (p < 0.005) as compared with control C. parvum‐treated mice. These data are consistent with the hypothesis that inflammation modulates miRNA expression in vivo and the alteration in specific miRNA under an inflammatory microenvironment, can be influenced by p53 (miR‐34b/c) and NO^? (29b/c).     

PTPRO-mediated autophagy prevents hepatosteatosis and tumorigenesis

Autophagy plays a critical role in the progression of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Protein tyrosine phosphatase receptor type O (PTPRO) was recently identified as a tumor suppressor, but little is known about its role in NASH. Here, we investigated the role of PTPRO-dependent autophagy in insulin resistance, lipid metabolism, and hepatocarcinogenesis. Wild-type (WT) and ptpro^−/− mice were fed a high-fat diet (HFD) for another 16 weeks after diethylnitrosamine (DEN) injection to induce NASH. Ptpro^−/− mice exhibited severe liver injury, insulin resistance, hepatosteatosis and autophagy deficiency compared with WT littermates. PTPRO deletion also promoted the induction of lipogenic target genes and decreases in β-oxidation-related genes. Increased activation of AKT and accumulation of cytoplasmic p53 was detected in ptpro^−/− mice, which in combination repressed autophagy. Intriguingly, hyperinsulinemia involving AKT activation was also exacerbated in HFD-fed mice due to PTPRO deletion. Activation of AKT induced stabilization of the MDMX/MDM2 heterocomplex, thus promoting p53 accumulation in the cytoplasm. Inhibition of AKT restored autophagy and p53 accumulation in hepatocytes, indicating that AKT acts upstream of p53. Due to hyperinsulinemia and autophagy deficiency, a HFD could aggravate steatohepatitis in ptpro^−/− mice. Importantly, the expression of PTPRO was much decreased in human steatohepatitis, which was associated with increased p62 accumulation. Together, these data indicate that PTPRO regulates insulin and lipid metabolism via the PI3K/Akt/MDM4/MDM2/P53 axis by affecting autophagy.