Nucleophagy contributes to genome stability though TOP2cc and nucleolar components degradation

ABSTRACTThe nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal, healthy cells maintain genome stability avoiding accumulation of nuclear damaged material is less understood. Here we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after Etoposide-induced DNA Double-Stranded Breaks. These nuclear buds and micronuclei co-localize with autophagic proteins BECN1 and LC3 and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and Type II DNA Topoisomerases (TOP2A and TOP2B), or nucleolar protein Fibrillarin, implying they are also targets of nucleophagy. We propose that a basal nucleophagy contributes to genome and nuclear stability and also in response to DNA damage and nucleolar stress.

Multifunctional thiosemicarbazones targeting sigma receptors: in vitro and in vivo antitumor activities in pancreatic cancer models

Purpose Association of the metal chelating portion of thiosemicarbazone with the cytotoxic activity of sigma-2 receptors appears a promising strategy for the treatment of pancreatic tumors. Here, we developed a novel sigma-2 receptor targeting thiosemicarbazone (FA4) that incorporates a moiety associated with lysosome destabilization and ROS increase in order to design more efficient antitumor agents. Methods The density of sigma receptors in pancreatic cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (mRNA expression of GRP78, ATF6, IRE1, PERK; ROS levels by MitoSOX and DCFDA-AM; JC-1 staining) induced by the thiosemicarbazones FA4, MLP44, PS3 and ACthio-1, were evaluated. The expression of autophagic proteins (ATG5, ATG7, ATG12, beclin, p62 and LC3-I) was also studied. In addition, the in vivo effect of FA4 in xenograft models with and without gemcitabine challenge was investigated. Results We found that FA4 exerted a more potent cytotoxicity than previously studied thiosemicarbazones (MLP44, PS3 and ACthio-1), which were found to display variable effects on the ER or the mitochondria-dependent pro-apoptotic axis. By contrast, FA4 activated pro-apoptotic pathways and decreased autophagy, except in MiaPaCa2 cells, in which autophagic proteins were expressed at lower levels and remained unmodified by FA4. FA4 treatment of PANC-1 xenografted mouse models, poorly responsive to conventional chemotherapy, significantly reduced tumor volumes and increased intratumor apoptosis compared to gemcitabine, with no signs of toxicity. Conclusions Our data indicate that FA4 exhibits encouraging activity in pancreatic cancer cells unresponsive to gemcitabine. These results warrant further investigation in patient-derived pancreatic cancers, and hold promise for the development of therapies that can more efficiently target the specific characteristics of individual tumor types.

TAT-Beclin1 Represses the Carcinogenesis of DUSP4-positive PTC by Enhancing Autophagy

Background: DUSP4 is a pro-tumorigenic molecule of papillary thyroid carcinoma (PTC). DUSP4 also exists as an autophagic regulator. Moreover, DUSP4, as a negative regulator of MAPK, can prevent Beclin1 from participating in autophagic response. This study aimed to explore whether TAT-Beclin1, a recombinant protein of Beclin1, could inhibit the tumorigenesis of DUSP4-positive PTC by regulating autophagy.Methods: First, we divided PTC cancer tissues into three groups according to DUSP4 expression levels by immunohistochemical analyses, and evaluated the relationship between the expression of autophagic proteins (Beclin1 and LC3II) and DUSP4 expression using Western blotting assays. After overexpression of DUSP4 by lentiviral transduction, the roles of TAT-Beclin1 on DUSP4-overexpressed PTC was detected.Results: Our results showed that the expression levels of autophagic proteins (Beclin1 and LC3II) increased with the increase of DUSP4 expression in PTC carcinomas. In PTC cells, DUSP4 overexpression-inhibited autophagic activity (including Beclin1 expression, LC3 conversion rate and LC3-puncta formation) and -promoted cell proliferation and migration were reversed by TAT-Beclin1 administration. In vivo assays also showed that DUSP4-overexpressed PTC cells had stronger tumorigenic ability and weaker autophagic activity, which was recovered by TAT-Beclin1 administration. Conclusions: TAT-Beclin1, as an autophagic promoter, could repress the carcinogenesis of DUSP4-positive PTC, which implies that the addition of TAT-Beclin1 may be determined through detecting the levels of DUSP4 in the treatment of PTC.

Autophagic Heterogeneity in Gastric Adenocarcinoma

Background and AimGastric/gastroesophageal junction (GEJ) adenocarcinoma is a heterogeneous disease, with various etiologies and with tumors encompassing a spectrum of histologic and molecular subtypes. “Autophagy” includes two related but distinct homeostatic processes that promote cell survival under adverse conditions, namely macro- and chaperone-mediated autophagy. There is increasing evidence of the roles autophagy may play in tumorigenesis.MethodsAutophagic pathways were examined in the context of the heterogeneity intrinsic to gastric/GEJ adenocarcinoma, utilizing immunohistochemistry targeting specific proteins within the pathways (p62, LAMP2A, LC3B). We examined whole sections of normal and dysplastic gastric mucosa, as well as a tissue microarray of adenocarcinomas.ResultsDysplastic gastric epithelium was marked by frequent nuclear p62 and aberrant LAMP2A expression compared to normal. Examining the pattern of LC3B/cytoplasmic p62 immuno-reactivity in gastric adenocarcinoma demonstrated a predominant pattern of LC3BHigh/p62High staining (56/86, 65.1%), which has been previously associated with active, but impaired macroautophagy. There were no statistically significant associations seen between LC3B/cytoplasmic p62 staining patterns with tumor grade, histotype, or approximated TCGA molecular subtype. LAMP2A and nuclear p62 and staining patterns were also heterogeneous across the cohort, but with no statistically significant associations seen. The prognostic significance of the three proteins was limited, however high nuclear p62 levels were associated with worse overall survival (log-rank p-value = 0.0396).ConclusionOur data demonstrate the dynamic nature of autophagic proteins in the gastric epithelium, and we expand the biological heterogeneity observed in gastric/GEJ adenocarcinoma to include autophagy.

Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

Methotrexate and theaflavin-3,3’ di gallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: An ex vivo approach with cultured human RA FLS

Background: Rheumatoid arthritis (RA) is characterized by inflammation mediated angiogenesis in synovial tissue, leading to apoptotic retardation and enhanced cell survival in synovial fibroblasts. Methotrexate (MTX) can reduce selective pro-inflammatory cytokines but unable to restore disrupted homeostasis between autophagy and apoptosis in fd-FLS.Objective: To evaluate the effect of black tea compound TF3 along with MTX upon fluid derived (fd)-FLS to induce apoptosis and inhibit autophagy through ER stress-mediated pathways.Methods: FLS sourced from synovial fluid (SF) of patients with RA (n=11) and osteoarthritis (OA) (n=10) were cultured following treatment with MTX/TF3 or in combination and underlying mechanisms were investigated. Extracellular inflammatory markers like CRP and cytokines (TNF-α, IL-6), angiogenic markers (VEGF, ANG-1) were quantified by ELISA. Cell viability of cultured fd-FLS was determined by MTT assay. fd-FLS treated with MTX/TF3 or combination of MTX(125nM) and TF3(10µM), followed by apoptosis measurement by flow cytometry. ER stress associated markers were quantified by RT-PCR (IRE1A and spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF1-α). Apoptotic (Bcl-2, Bax, and Caspases) and autophagic proteins (Beclin1, LC3b and p62) were quantified by immunoblot study. Results: MTX and TF3 both in single doses (IC25) could down-regulate the levels of pro-inflammatory and angiogenic markers. Combination treatment modulated ER stress response and blocked the auto-phagmosomal proteins in fd-FLS and induced apoptosis.Conclusion: Disruption in homeostasis between apoptosis and autophagy might be an underlying phenomenon in the progression of pathophysiology in fd-FLS. The combined administration of MTX and TF3 successfully balanced the homeostasis by inducing apoptosis.

Autophagy, an accomplice or antagonist of drug resistance in HCC?

Hepatocellular carcinoma (HCC) is a highly lethal malignancy characterized by poor prognosis and a low 5-year survival rate. Drug treatment is proving to be effective in anti-HCC. However, only a small number of HCC patients exhibit sensitive responses, and drug resistance occurs frequently in advanced patients. Autophagy, an evolutionary process responsible for the degradation of cellular substances, is closely associated with the acquisition and maintenance of drug resistance for HCC. This review focuses on autophagic proteins and explores the intricate relationship between autophagy and cancer stem cells, tumor-derived exosomes, and noncoding RNA. Clinical trials involved in autophagy inhibition combined with anticancer drugs are also concerned.

MiR-30a sensitized lung cancer against neoadjuvant chemotherapy by depressing autophagy

Objective This study was aimed at exploring whether miR-30a enhanced sensitivity of non-small-cell lung cancer (NSCLC) cells against neoadjuvant chemotherapy through an autophagy-dependent way. Methods We totally recruited 304 NSCLC patients who have underwent chemotherapy, as well as 185 NSCLC patients who did not receive chemotherapy. NSCLC cell lines (i.e. H1299 and H460) were also purchased, and they were transfected by miR-30a mimic/inhibitor. Furthermore, cisplatin (DDP)/pemetrexed (PEM) resistance of NSCLC cells was assessed utilizing MTT assay, and autophagic proteins isolated from NSCLC tissues and cells were quantitated by western blotting. Results Lowly expressed miR-30a was reflective of lymph node metastasis, advanced TNM stage and poor 5-year survival among NSCLC patients treated by neoadjuvant chemotherapy (i.e. combined treatment of DDP and PEM) (P < 0.05). Moreover, DDP combined with PEM attenuated viability and proliferation, but, on the contrary, promoted autophagy of H1299 and H460 cell lines (P < 0.05). However, miR-30a undermined resistance of NSCLC cells against DDP and PEM (P < 0.05), and it suppressed DDP/PEM-induced autophagy and promoted DDP/PEM-triggered apoptosis of NSCLC cells (P < 0.05). Conclusions Intentionally elevating miR-30a expression was conducive to improving NSCLC prognosis after neoadjuvant chemotherapy, for its depressing drug-caused autophagy and resistance.

Sulforaphane Inhibits Osteoclastogenesis via Suppression of the Autophagic Pathway

Previous studies have demonstrated that sulforaphane (SFN) is a promising agent against osteoclastic bone destruction. However, the mechanism underlying its anti-osteoclastogenic activity is still unclear. Herein, for the first time, we explored the potential role of autophagy in SFN-mediated anti-osteoclastogenesis in vitro and in vivo. We established an osteoclastogenesis model using receptor activator of nuclear factor kappa-β ligand (RANKL)-induced RAW264.7 cells and bone marrow macrophages (BMMs). Tartrate-resistant acid phosphatase (TRAP) staining showed the formation of osteoclasts. We observed autophagosomes by transmission electron microscopy (TEM). In vitro, we found that SFN inhibited osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the SFN (0) group vs. 20.33 ± 1.45 in the SFN (1 μM) group vs. 13.00 ± 1.00 in the SFN (2.5 μM) group vs. 6.66 ± 1.20 in the SFN (2.5 μM) group), decreased the number of autophagosomes, and suppressed the accumulation of several autophagic proteins in osteoclast precursors. The activation of autophagy by rapamycin (RAP) almost reversed the SFN-elicited anti-osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the control group vs. 13.00 ± 1.00 in the SFN group vs. 17.33 ± 0.33 in the SFN+RAP group). Furthermore, Western blot (WB) analysis revealed that SFN inhibited the phosphorylation of c-Jun N-terminal kinase (JNK). The JNK activator anisomycin significantly promoted autophagy, whereas the inhibitor SP600125 markedly suppressed autophagic activation in pre-osteoclasts. Microcomputed tomography (CT), immunohistochemistry (IHC), and immunofluorescence (IF) were used to analyze the results in vivo. Consistent with the in vitro results, we found that the administration of SFN could decrease the number of osteoclasts and the expression of autophagic light chain 3 (LC3) and protect against lipopolysaccharide (LPS)-induced calvarial erosion. Our findings highlight autophagy as a crucial mechanism of SFN-mediated anti-osteoclastogenesis and show that the JNK signaling pathway participates in this process.