Overexpressing STAMP2 attenuates diabetic renal injuries via upregulating autophagy in diabetic rats.

Diabetic nephropathy (DN) is one of the most serious and major renal complications of diabetes. Previously, Six-transmembrane Protein of Prostate 2 (STAMP2) was reported to contribute to nutritional stress. The purpose of this study is to investigate whether overexpression of STAMP2 attenuates diabetic renal injuries in DN rats. We induced the DN rat model by high-fat diet and low-dose streptozotocin and evaluated the metabolite and urine albumin/creatinine. Recombinant adeno-associated virus vectors were injected for overexpression of STAMP2. Pathophysiologic and ultrastructure features of DN by histochemical stain and transmission electron microscope, autophagy-related proteins and signaling pathway by western blotting were assessed. We found the expression of STAMP2 was decreased and autophagy was blunted in DN rat kidneys. Overexpressing STAMP2 significantly ameliorated metabolic disturbance, insulin resistance, and specifically restoring diabetic renal injury. Furthermore, overexpressing STAMP2 improved the autophagy deficiency in DN rats, as revealed by changes in the expressions of Beclin1, p62, and LC3. Furthermore, STAMP2 overexpressing promoted autophagy by inhibiting the mTOR and activating the AMPK/SIRT1 signaling pathway. Our results suggested that STAMP2 overexpression attenuated renal injuries via upregulating autophagy in DN rats. STAMP2 overexpressing promoted autophagy may been involved with inhibition of the mTOR/ULK1 and activation of the AMPK/SIRT1 signaling pathway.

MicroRNA-3473b regulates the expression of TREM2/ULK1 and inhibits autophagy in inflammatory pathogenesis of Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disease characterized by selective loss of dopaminergic (DA) neurons in the midbrain. The regulatory role of a variety of microRNAs in PD has been confirmed, and our study is the first to demonstrate that miR-3473b is involved in the regulation of PD. In vitro, an miR-3473b inhibitor can inhibit the secretion of inflammatory factors (TNF-α and IL-1ß) in moues microglia cell line (BV2) cells induced by lipopolysaccharide (LPS) and promote autophagy in BV2 cells. In vivo, miR-3473b antagomir can inhibit the activation of substantia nigra pars compacta (SNpc) microglia of C57BL/6 mice induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and promote autophagy. Deletion of TREM2, one of the most highly expressed receptors in microglia, leads to the occurrence and development of PD. ULK1 is a component of the Atg1 complex. Deletion of ULK1 aggravates the pathological reaction of PD. TREM2 and ULK1 are predicted potential targets of miR-3473b by Targetscan. Then, the results of our experiments indicate that transfection with a miR-3473b mimic can inhibit the expression of TREM2 and ULK1. Data from a double luciferase experiment indicate that the 3'-UTR of TREM2, but not ULK1, is the direct target of miR-3473b. Then we aim to investigate the regulation of TREM2 and ULK1 in PD. We found that the expression of p-ULK1 was significantly increased via up-regulation of TREM2. The increased expression of p-ULK1 can promote autophagy and inhibit the expression of inflammatory factors. The regulation of ULK1 by miR-3473b may be accomplished indirectly through TREM2. Thus, miR-3473b may regulate the secretion of proinflammatory mediators by targeting TREM2/ULK1 expression to regulate the role of autophagy in the pathogenesis of inflammation in Parkinson's disease, suggesting that mir-3473b may be a potential therapeutic target to regulate the inflammatory response in PD.

ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia.

BACKGROUND: In acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with a dismal outcome. Although uncoordinated 51-like kinase 1 (ULK1), which plays a central role in the autophagy pathway, has emerged as a novel therapeutic target for various cancers, its role in FLT3-ITD AML remains elusive. In this study, we evaluated the effects of ULK1 inhibition on leukemia cell death in FLT3-ITD AML. METHOD: We evaluated ULK1 expression and the levels of apoptosis and autophagy following ULK1 inhibition in FLT3-ITD AML cell lines and investigated the mechanism underlying apoptosis induced by ULK1 inhibition. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc). RESULTS: FLT3-ITD AML cells showed significantly higher ULK1 expression than FLT3-wild-type (WT) AML cells. Two ULK1 inhibitors, MRT 68921 and SBI-0206965, induced apoptosis in FLT3-ITD AML cells, with relatively minimal effects on FLT3-WT AML cells and normal CD34-positive cells. Apoptosis induction by ULK1 inhibition was associated with caspase pathway activation. Interestingly, ULK1 inhibition paradoxically also induced autophagy, showing synergistic interaction with autophagy inhibitors. Hence, autophagy may act as a prosurvival mechanism in FLT3-ITD AML cells. FLT3-ITD protein degradation and inhibition of the ERK, AKT, and STAT5 pathways were also observed in FLT3-ITD AML cells following treatment with ULK1 inhibitors. CONCLUSION: ULK1 is a viable drug target and ULK1 inhibition may represent a promising therapeutic strategy against FLT3-ITD AML.

Combination of ULK1 and LC3B improve prognosis assessment of hepatocellular carcinoma.

BACKGROUND: Autophagy involves in both prevention and promotion in cancer, and its role probably changed during tumor development. Defined the dynamic function of autophagy in cancer may advance precision diagnostics, treatment, and guide drug design. Autophagy related protein ULK1 is key regulator of autophagy, and its role in hepatocellular carcinoma (HCC) was still unclear. This study aims to investigate ULK1's capacity along with other autophagic markers in predicting prognosis of HCC and explore position of these biomarkers in dynamic function of autophagy during HCC progression. METHODS: The expression of ULK1 and other autophagic marker (LC3B) were test by Tissue microarray-based immunohistochemistry in 156 operable HCC patients. Survival analysis and correlation analysis were used to analysis influence of ULK1 and combined biomarker on clinical characteristics and prognosis. RESULTS: The expression level of ULK1 was not related to all clinicopathological features, however, high expression of the ULK1 as well as LC3B overexpression suggested large tumor size (P=0.035), high levels of serum AFP (P=0.049), more frequency of node metastasis (P=0.015), later TNM stage (P=0.009). Survival analysis showed that ULK1 expression were negatively correlated with PFS rather than OS in HCC patients (P=0.021), while LC3B were suggested to be negatively related with patients' PFS, However, Simultaneous high expression of ULK1 and LC3B had a poorer 5-year overall survival (OS) rate (P=0.002) and shorter 5-year progression free survival (PFS)(P=0.003), Further multivariate analysis revealed that the two combined biomarkers were independent factors to predict the prognosis of OS and PFS in all patients, while ULK1 alone or LC3B alone were only an independent predict factor for OS or PFS respectively. CONCLUSION: ULK1 were demonstrated to be an important prognostic factor for HCC patient, and it combined LC3B would improve prognosis assessment of the patients. Combined autophagic biomarkers would better represent dynamic stage of autophagy and It might provide a potential therapeutic way that how to interfere autophagy in HCC.

Sirtuin 6 plays an oncogenic role and induces cell autophagy in esophageal cancer cells.

Sirtuin 6, a member of sirtuin family, is generally regarded as a tumor suppressor as it participates in suppressing hypoxia-inducible factor 1α and MYC transcription activity by deacetylating H3K9 (histone H3 lysine 9) and H3K56 (histone H3 lysine) at promoters of target genes, leading to the aerobic glycolysis inhibition and cell growth suppression. However, its expression has recently been reported to be highly elevated in a series of tumors, including prostate cancer, breast cancer, and non-small cell lung cancer, indicating that sirtuin 6 plays dual roles in tumorigenicity in a cell/tumor type-specific manner. To our knowledge, the biological roles of sirtuin 6 in esophageal cancer cells have still been underestimated. In the study, data from quantitative reverse transcriptase polymerase chain reaction-based assays and immunohistochemical assays revealed that sirtuin 6 was remarkably overexpressed in esophageal squamous tumor tissues. Moreover, its upregulation was closely related with clinical features, such as gender, pathology, tumor-node-metastasis, and cell differentiation. Subsequently, the biological tests showed that it promoted cell proliferation and induced the expression of Bcl2, a key anti-apoptotic factor, in esophageal carcinoma cells. Moreover, using the ratio of LC3II/I, a widely recognized autophagy biomarker, we showed that it apparently induced cell autophagy, which was further confirmed by the autophagy flux assays. In addition, results from western blotting assays and immunoprecipitation assays displayed that sirtuin 6 specifically interacted with ULK1 and positively regulated its activity by inhibiting its upstream factor mammalian target of rapamycin activity. In summary, our studies shed insights into the crucial functions of sirtuin 6 in esophageal carcinoma cells and provide evidence supporting sirtuin 6-based personalized therapies in esophageal carcinoma cell patients.

Silencing of NRAGE induces autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells.

NRAGE has been reported to be overexpressed in cancer cells, especially in lung cancer cells. To determine the role of NRAGE in non-small-cell lung cancer cells, we investigated the effects of NRAGE on autophagy in non-small-cell lung cancer cells. Human A549 and H1299 cells were transfected with NRAGE-specific small-interfering RNA. The Cell Counting Kit-8 and plate clone assay showed that downregulation of NRAGE could induce the proliferation in A549 and H1299 cells. In addition, our data suggested that downregulation of NRAGE enhances autophagosome formation by immunofluorescence. We found that knockdown of NRAGE induced autophagy, together with downregulation of P62 and upregulation of LC3-II protein. Furthermore, to elucidate the mechanism of NRAGE in suppressing autophagy, the protein expressions of AMPK, Ulk1, and Atg13 were assessed. Collectively, these results demonstrate the effective anti-autophagic of NRAGE in non-small-cell lung cancer cells through AMPK/Ulk1/Atg13 autophagy signaling pathways. Therefore, NRAGE could be used as a potential therapeutic target for lung cancer.

Crocetin shifts autophagic cell survival to death of breast cancer cells in chemotherapy.

The chemotherapy with fluorouracil is not always effective, in which some breast cancer cells may survive the fluorouracil treatment through enhanced autophagy. Crocetin is the major constituent of saffron, a Chinese traditional herb, which has recently found to have multiple pharmacological effects, including anticancer. However, the effects of Crocetin on the outcome of fluorouracil therapy for breast cancer have not been studied. Here, we showed that fluorouracil treatment inhibited the growth of breast cancer cells, in either a Cell Counting Kit-8 assay or an MTT assay. Inhibition of autophagy further suppressed breast cancer cell growth, suggesting that the breast cancer cells increased autophagic cell survival during fluorouracil treatment. However, Crocetin significantly increased the suppressive effects of fluorouracil on breast cancer cell growth, without affecting either cell apoptosis or autophagy. Inhibition of autophagy at the presence of Crocetin partially abolished the suppressive effects on breast cancer cell growth, suggesting that Crocetin may increase autophagic cell death in fluorouracil-treated breast cancer cells. Furthermore, Crocetin decreased Beclin-1 levels but increased ATG1 levels in fluorouracil-treated breast cancer cells. Together, these data suggest that Crocetin may shift autophagic cell survival to autophagic cell death in fluorouracil-treated breast cancer cells, possibly through modulation of the expression of ATG1 and Beclin-1.

Regulation of ULK1 Expression and Autophagy by STAT1.

Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy.