Differences in Levels of Mitochondrial DNA Content at Various Stages of Canine Myxomatous Mitral Valve Disease.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in small-breed dogs, often leading to heart failure. Oxidative stress in MMVD can harm mitochondria, decreasing their DNA content. This study assesses dogs' oxidative stress and mitochondrial DNA at different MMVD stages. Fifty-five small-breed dogs were categorized into four groups, including: A-healthy (n = 15); B-subclinical (n = 15); C-heart failure (n = 15); and D-end-stage MMVD (n = 10). Serum malondialdehyde (MDA) and mitochondrial DNA in peripheral blood were analyzed. Quantitative real-time PCR measured mitochondrial DNA, and PCR data were analyzed via the fold-change Ct method. Serum MDA levels were assessed using competitive high-performance liquid chromatography (HPLC). Mitochondrial DNA was significantly lower in group B (-0.89 ± 2.82) than in group A (1.50 ± 2.01), but significantly higher in groups C (2.02 ± 1.44) and D (2.77 ± 1.76) than B. MDA levels were notably elevated in groups B (19.07 ± 11.87 µg/mL), C (23.41 ± 12.87 µg/mL), and D (19.72 ± 16.81 µg/mL) in comparison to group A (9.37 ± 4.67 µg/mL). Nevertheless, this observed difference did not reach statistical significance. It is noteworthy that mitochondrial DNA content experiences a decline during the subclinical stage but undergoes an increase in cases of heart failure. Concurrently, oxidative stress exhibits an upward trend in dogs with MMVD. These findings collectively suggest a potential association between mitochondrial DNA, oxidative stress, and the progression of MMVD in small-breed dogs.

Activating Mutation in the Receptor Tyrosine Kinase FLT3 with Clinicopathological Relevance in Canine Mast Cell Tumors.

Recent research has focused on the receptor tyrosine kinase (RTK) KIT which is involved in the pathogenesis of canine mast cell tumors (MCT). However, the role of other RTKs in this neoplasm remains unclear. The present study aimed to determine the frequency of FLT3 mutations and to evaluate the mutational status and clinicopathological relevance of canine MCT patients. There were a total of 20 cases that were cytologically and histopathological diagnosed as canine MCTs; genomic polymerase chain reaction (PCR) and Sanger sequencing were used to identify mutations. For the juxtamembrane (JM) domain, the FLT3 14/15 primer pair was used to investigate exon 14/15 loci. Based on genomic PCR amplification of exon 14/15 and 20 of the FLT3 gene and Sanger sequencing of 20 cases of canine MCTs, the overall frequency of FLT3 mutation in canine MCTs was 75%. The majority of FLT3 mutations (70%) were internal tandem duplications (ITD) of the JM domain, while one case arose from deletion mutations of the tyrosine kinase domain (TKD). However, double mutations were not observed in this study. Furthermore, there is also clinicopathological relevance to MCT dogs carrying FLT3-ITD mutations, showing a tendency toward leukocytosis due to neutrophilia, and resembling human acute myeloid leukemia (AML) with FLT3-ITD mutations. A subset of MCTs with FLT3-ITD mutations, showing an enhanced signal of phosphorylated ERK1/2 identified by immunoblotting, suggests that an activating mutation may be driven by a distinct signal of the ERK pathway. Our results indicate that FLT3-ITD mutation is an oncogenic driver of canine MCTs, and that it shares some clinicopathologic features with human AML. These findings may offer new opportunities for further studies on canine mast cell tumorigenesis and a novel therapeutic target for canine MCT cases harboring FLT3-ITD mutations.

Impact of serum C-reactive protein level as a biomarker of cancer dissemination in canine lymphoid neoplasia.

Background and Aim: C-reactive protein (CRP) is a highly sensitive but non-specific acute phase protein that has been widely used to predict the biological behavior of patients with cancer. This study aimed to examine the significance of the serum CRP biomarker in predicting the prognosis of dogs with lymphoma. Materials and Methods: Blood samples (5 mL) were collected from 34 lymphoma dogs and control healthy dogs. Canine lymphoma clinical staging was classified using the World Health Organization (WHO) criteria. All lymphoma dogs were reclassified into two groups based on the disease stage. Stages IV and V were designated as advanced stages, and Stages I-III were designated as other stages. The serum CRP level was then determined using a commercial canine CRP fluorescent immunoassay kit and routine hematological and biochemical analyses. C-reactive protein levels, circulating inflammatory parameters, such as neutrophil-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, and platelet-to-lymphocyte ratio, and albumin levels were compared between advanced stages (IV and V) and Stages I to III using Mann-Whitney U tests. Receiver operating characteristic (ROC) curves were also generated to determine the cutoff value, diagnostic sensitivity, and specificity of the CRP level. Results: A prospective study identified 34 dogs recently diagnosed with canine lymphoma. C-reactive protein levels were significantly higher in lymphoma dogs in advanced stages (IV and V) than in lymphoma dogs in Stages I-III. According to the ROC curve analysis, a CRP cutoff level of 54.1 mg/L indicates advanced-stage canine lymphoma, which can be used as a biomarker to predict cancer dissemination. Conclusion: Serum CRP concentrations can assist clinical decision-making on the WHO stage in lymphoma dogs in clinical applications. The limitations of this study include a small number of lymphomas and no survival analysis.

Novel PRMT5-mediated arginine methylations of HSP90A are essential for maintenance of HSP90A function in NDRG2low ATL and various cancer cells.

N-myc downstream-regulated gene 2 (NDRG2) as a tumor suppressor is frequently downregulated in human T-lymphotropic retrovirus (HTLV-1)-infected adult T-cell leukemia (ATL) and variety of cancers, and negatively regulates PI3K signaling pathways through dephosphorylation of PTEN with protein phosphatase 2A (PP2A). We recently identified that protein arginine methyltransferase 5 (PRMT5) is one of novel NDRG2 binding proteins and the knockdown of PRMT5 induces cell apoptosis with degradation of several signaling molecules. To investigate how the apoptosis is induced by the knockdown PRMT5 expression, heat shock protein 90 alpha (HSP90A) was identified as a binding protein for NDRG2 or PRMT5 by immunoprecipitation-mass analysis. NDRG2/PP2A complex inhibited arginine methyltransferase activity of PRMT5 through dephosphorylation at Serine 335 (S335); however, in NDRG2low ATL-related cells, highly phosphorylated PRMT5 at S335 was mainly localized in cytoplasm with binding to HSP90A, resulting in enhancing arginine-methylation at the middle domain (R345 and R386). Since knockdown of PRMT5 expression or forced expression of HSP90A with alanine replacement of R345 or R386 induced apoptosis with the degradation of client proteins in NDRG2low ATL-related and other cancer cells, we here identified that the novel arginine methylations of HSP90A are essential for maintenance of its function in NDRG2low ATL and other cancer cells.

Activation of EVI1 transcription by the LEF1/ß-catenin complex with p53-alteration in myeloid blast crisis of chronic myeloid leukemia.

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/ß-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of ß-catenin is partly dependent on BCR-ABL expression through enhanced GSK3ß phosphorylation, and EVI1 expression is directly enhanced by the LEF1/ß-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on ß-catenin activation through GSK3ß phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/ß-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression.

The loss of NDRG2 expression improves depressive behavior through increased phosphorylation of GSK3ß.

N-myc downstream-regulated gene 2 (NDRG2) is one of the important stress-inducible genes and plays a critical role in negatively regulating PI3K/AKT signaling during hypoxia and inflammation. Through recruitment of PP2A phosphatase, NDRG2 maintains the dephosphorylated status of PTEN to suppress excessive PI3K/AKT signaling, and loss of NDRG2 expression is frequently seen in various types of cancer with enhanced activation of PI3K/AKT signaling. Because NDRG2 is highly expressed in the nervous system, we investigated whether NDRG2 plays a functional role in the nervous system using Ndrg2-deficient mice. Ndrg2-deficient mice do not display any gross abnormalities in the nervous system, but they have a diminished behavioral response associated with anxiety. Ndrg2-deficient mice exhibited decreased immobility and increased head-dipping and rearing behavior in two behavioral models, indicating an improvement of emotional anxiety-like behavior. Moreover, treatment of wild-type mice with the antidepressant drug imipramine reduced the expression of Ndrg2 in the frontal cortex, which was due to the degradation of HIF-1α through reduced expression of HSP90 protein. Furthermore, we found that the down-regulation of Ndrg2 in Ndrg2-deficient mice and imipramine treatment improved mood behavior with enhanced phosphorylation of GSK3ß through activation of PI3K/AKT signaling, suggesting that the expression level of NDRG2 has a causal influence on mood-related phenotypes. Collectively, these results suggest that NDRG2 may be a potential target for mood disorders such as depression and anxiety.

Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers.

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.