HYOU1 facilitates proliferation, invasion and glycolysis of papillary thyroid cancer via stabilizing LDHB mRNA.

HYOU1 is upregulated in many kinds of cancer cells, and its high expression is associated with tumour invasiveness and poor prognosis. However, the role of HYOU1 in papillary thyroid cancer (PTC) development and progression remains to be elucidated. Here, we reported that HYOU1 was highly expressed in human PTC and associated with poor prognosis. HYOU1 silencing suppressed the proliferation, migration and invasion of PTC cells. Mechanistic analyses showed that HYOU1 silencing promoted oxidative phosphorylation while inhibited aerobic glycolysis via downregulating LDHB at the posttranscriptional level. We further confirmed that the 3'UTR of LDHB mRNA is the indirect target of HYOU1 silencing and HYOU1 silencing increased miR-375-3p levels. While LDHB overexpression significantly suppressed the inhibitory effects of HYOU1 silencing on aerobic glycolysis, proliferation, migration and invasion in PTC cells. Taken together, our findings suggest that HYOU1 promotes glycolysis and malignant progression in PTC cells via upregulating LDHB expression, providing a potential target for developing novel anticancer agents.

p53-dependent transcriptional suppression of BAG3 protects cells against metabolic stress via facilitation of p53 accumulation.

Solid tumour frequently undergoes metabolic stress during tumour development because of inadequate blood supply and the high nutrient expenditure. p53 is activated by glucose limitation and maintains cell survival via triggering metabolic checkpoint. However, the exact downstream contributors are not completely identified. BAG3 is a cochaperone with multiple cellular functions and is implicated in metabolic reprogramming of pancreatic cancer cells. The current study demonstrated that glucose limitation transcriptionally suppressed BAG3 expression in a p53-dependent manner. Importantly, hinderance of its down-regulation compromised cellular adaptation to metabolic stress triggered by glucose insufficiency, supporting that BAG3 might be one of p53 downstream contributors for cellular adaptation to metabolic stress. Our data showed that ectopic BAG3 expression suppressed p53 accumulation via direct interaction under metabolic stress. Thereby, the current study highlights the significance of p53-mediated BAG3 suppression in cellular adaptation to metabolic stress via facilitating p53 accumulation.

AUF1 modulates TGF-ß signal in renal tubular epithelial cells via post-transcriptional regulation of Nedd4L expression.

Posttranscriptional regulation process plays important roles in renal disease pathogenesis. AU-rich element RNA-binding protein (AUF1) interacts with and destabilizes mRNAs containing AU-rich elements (AREs) in their 3'UTR. The current study demonstrated that AUF1 was increased in unilateral ureteral obstruction (UUO) animal models. While proliferation and migration of HK2 cells was unaltered by AUF1 downregulation under normal condition, proliferative inhibition and migratory promotion mediated by TGF-ß was significantly compromised. Mechanically, AUF1 downregulation decreased phosphorylated Smad2/3 via increasing their E3 ligase Nedd4L at the posttranscriptional level. In addition, the current study identified Nedd4L as a previously unreported target of AUF1. AUF1 regulates Nedd4L expression at the posttranscriptional level by interaction with AREs in the 3'UTR of the Nedd4L mRNA. Collectively, the current study indicates that AUF1 might be a potential player in renal tubulointerstitial fibrosis through modulation of TGF-ß signal transduction via posttranscriptional regulation of Nedd4L.

Sestrin 2 protects against metabolic stress in a p53-independent manner.

Glucose limitation activates p53, which functions as an adaptive response to maintain cell survival. However, p53 is frequently deleted or mutated in a variety of tumors, while most cancer cells can acclimatize themselves to metabolically unfavorable surrounding, indicating that alternative mechanisms other than p53 transactivation underly adaptive response of cancer cells with p53 deletion or mutation to metabolically hostile environment. Sestrin 2 (SESN2) is a p53 downstream target, which plays a protective role against various stressful stimuli, such as genotoxic, energetic, and oxidative stress. In the current study, we demonstrated that SESN2 transcript was stabilized by glucose limitation at the posttranscriptional level irrespective of p53 status. Importantly, SESN2 also protected cells from metabolic stress triggered by glucose limitation in a p53-independent manner. Our data indicated that stabilization of SESN2 transcript might be an alternative adaptive response to metabolic stress other than p53 activation. Thereby, the current study highlights the significance of stabilization of SESN2 transcript in adaptation of cells with p53 deletion or mutation to metabolic stress.

Advanced glycation end products promote proliferation and suppress autophagy via reduction of Cathepsin D in rat vascular smooth muscle cells.

Autophagy is closely involved in vascular smooth muscle cell (VSMC) function, but little is known about the association between advanced glycation end products (AGEs) and autophagy and its role in AGEs-induced proliferation and migration of VSMCs. The current study investigated the effects of AGEs on the phenotypic modulation and autophagy of VSMCs, as well as the potential underlying mechanisms. Primary rat VSMCs were treated with bovine serum albumin or AGEs. Cell proliferation was detected by MTT assay, real-time cell analyzer and EdU incorporation. Cell cycle was analyzed by Hoechst staining and flow cytometry. The migration of VSMCs was detected by wound-healing assay and transwell migration assay. LC3 transition and p62 accumulation were detected using Western blotting. Acidic vacuoles were measured using AO and MDC staining. Cathepsin D (CatD) was transduced to VSMCs via lentiviral vectors. AGEs enhanced proliferation and migration of primary rat VSMC in a time-dependent manner. AGEs significantly increased LC3-II transition and p62 expression, as well as accumulation of acidic vacuole, which was not further increased by bafilomycin A1. AGEs decreased CatD expression in a time-dependent pattern, and overexpression of CatD prohibited autophagy attenuation mediated by AGEs. CatD overexpression suppressed AGEs-induced proliferation of VSMCs. Nevertheless, CatD exhibited no effects on AGEs-induced migration of VSMCs. AGEs promote proliferation of VSMCs and suppress autophagy, at least in part via CatD reduction.

BAG3 is upregulated by c-Jun and stabilizes JunD.

BAG3 plays a regulatory role in a number of cellular processes, including cell proliferation, apoptosis, adhesion and migration, epithelial-mesenchymal transition (EMT), autophagy activation, and virus infection. The AP-1 transcription factors are implicated in a variety of important biological processes including cell differentiation, proliferation, apoptosis and oncogenesis. Recently, it has been reported that AP-1 protein c-Jun inhibits autophagy and enhances apoptotic cell death mediated by starvation. However, the molecular mechanisms remain unclear. For the first time, the current study demonstrated that serum starvation downregulated BAG3 at the transcriptional level via c-Jun. In addition, the current study reported that BAG3 stabilized JunD mRNA, which was, at least in part, responsible for the promotion of serum starvation mediated-growth inhibition by BAG3.

Implication of Nrf2 and ATF4 in differential induction of CHOP by proteasome inhibition in thyroid cancer cells.

Proteasome inhibition may cause endoplasmic reticulum (ER) stress, which has been reported to be implicated in the antitumoral effects of proteasome inhibitors. CCAAT/enhancer-binding protein homologous protein (CHOP) is induced by a variety of adverse physiological conditions including ER stress and is involved in apoptosis. We have reported that distinct induction of CHOP contributes to the responsiveness of thyroid cancer cells to proteasome inhibitors. However, the mechanism underlying differential induction of CHOP by proteasome inhibitors in thyroid cancer cells has not been well characterized. In the current study, we characterized that proteasome inhibition primarily activated the amino acid response element 1 (AARE1) on the CHOP promoter. We also demonstrated that although proteasome inhibition caused similar accumulation of activating transcription factor 4 (ATF4) in a panel of thyroid cancer cells, distinct amounts of ATF4 were recruited to the AARE1 element of CHOP promoter. In addition, we demonstrated that NF-E2-related factor 2 (Nrf2) was also implicated in the induction of CHOP by precluding the binding of ATF4 to the CHOP promoter. This study highlights the molecular mechanisms by which ATF4 and Nrf2 can control CHOP induction in thyroid cancer cells by proteasome inhibition.

Involvement of JNK and NF-κB pathways in lipopolysaccharide (LPS)-induced BAG3 expression in human monocytic cells.

Lipopolysaccharide (LPS) is an outer-membrane glycolipid component of Gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. Bcl-2-associated athanogene 3 (BAG3) is a survival protein that has been shown to be stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, proteasome inhibition, and human immunodeficiency virus 1 (HIV-1) infection. In addition, BAG3 regulates replication of Varicella-Zoster Virus (VZV) and Herpes Simplex Virus (HSV) replication, suggesting that BAG3 could participate in the host response to infection. In the current study, we found that LPS increased the expression of BAG3 in a dose- and time-dependent manner. Actinomycin D completely blocked the LPS-induced BAG3 accumulation, as well as LPS activated the proximal promoter of BAG3 gene, supported that the induction by LPS occurred at the level of gene transcription. LPS-induced BAG3 expression was blocked by JNK or NF-κB inhibition, suggesting that JNK and NF-κB pathways participated in BAG3 induction by LPS. In addition, we also found that induction of BAG3 was implicated in monocytic cell adhesion to extracellular matrix induced by LPS. Overall, the data support that BAG3 is induced by LPS via JNK and NF-κB-dependent signals, and involved in monocytic cell-extracellular matrix interaction, suggesting that BAG3 may have a role in the host response to LPS stimulation.

Resveratrol protects leukemic cells against cytotoxicity induced by proteasome inhibitors via induction of FOXO1 and p27Kip1.

BACKGROUND: It was reported recently that resveratrol could sensitize a number of cancer cells to the antitumoral effects of some conventional chemotherapy drugs. The current study was designed to investigate whether resveratrol could sensitize leukemic cells to proteasome inhibitors. METHODS: Leukemic cells were treated with MG132 alone or in combination with resveratrol. Cell viability was investigated using MTT assay, and induction of apoptosis and cell cycle distribution was measured using flow cytometry. Western blot and real-time RT-PCR were used to investigate the expression of FOXO1 and p27Kip1. CHIP was performed to investigate the binding of FOXO1 to the p27 Kip1 promoter. RESULTS: Resveratrol strongly reduced cytotoxic activities of proteasome inhibitors against leukemic cells. MG132 in combination with resveratrol caused cell cycle blockade at G1/S transition via p27Kip1 accumulation. Knockdown of p27Kip1 using siRNA dramatically attenuated the protective effects of resveratrol on cytotoxic actions of proteasome inhibitors against leukemic cells. Resveratrol induced FOXO1 expression at the transcriptional level, while MG132 increased nuclear distribution of FOXO1. MG132 in combination with resveratrol caused synergistic induction of p27Kip1 through increased recruitment of FOXO1 on the p27Kip1 promoter. CONCLUSIONS: Resveratrol may have the potential to negate the cytotoxic effects of proteasome inhibitors via regulation of FOXO1 transcriptional activity and accumulation of p27Kip1.

AUF1 Promotes Proliferation and Invasion of Thyroid Cancer via Downregulation of ZBTB2 and Subsequent TRIM58.

The pathogenesis of papillary thyroid cancer (PTC), the most common type of thyroid cancer, is not yet fully understood. This limits the therapeutic options for approximately 7% of invasive PTC patients. The critical role of AUF1 in the progression of thyroid cancer was first reported in 2009, however, its molecular mechanism remained unclear. Our study used CRISPR/Cas 9 system to knockdown AUF1 in IHH4 and TPC1 cells. We noticed that the expression of TRIM58 and ZBTB2 were increased in the AUF1 knockdown IHH4 and TPC1 cells. When TRIM58 and ZBTB2 were inhibited by small hairpin RNAs (shRNAs) against TRIM58 (shTRIM58) and ZBTB2 (shZBTB2), respectively, the proliferation, migration, and invasion ability of the AUF1-knockdown IHH4 and TPC1 cells were increased. In addition, two ZBTB2 binding sites (-719~-709 and -677~-668) on TRIM58 promoter and two AUF1 binding sites (1250-1256 and 1258-1265) on ZBTB2 3'-UTR were identified. These results suggested that AUF1 affecting thyroid cancer cells via regulating the expression of ZBTB2 and TRIM58.