Dongqin Yang

Induction of autophagy and senescence by knockdown of ROC1 E3 ubiquitin ligase to suppress the growth of liver cancer cells

Regulator of Cullins-1 (ROC1) or RING box protein-1 (RBX1) is an essential RING component of Cullin-RING ligase (CRL). Our previous studies showed that ROC1 is required for the growth of several cancer cell lines while ROC1 siRNA silencing inactivates CRL, leading to cell cycle arrest, cell senescence and/or apoptosis. However, it is completely unknown whether ROC1 knockdown triggers autophagic response by inactivating CRL. Moreover, the role of ROC1 in liver cancer remains elusive. In this study, we reported that ROC1 knockdown significantly inhibited the growth of liver cancer cells by sequentially and independently inducing autophagy and p21-dependent cell senescence. Mechanism analysis revealed that ROC1 silencing triggered autophagy by inhibition of mammalian target of rapamycin (mTOR) activity due to accumulation of mTOR-inhibitory protein Deptor, a substrate of CRL. Consistently, Deptor knockdown significantly blocked autophagy response upon ROC1 silencing. Biologically, autophagy response upon ROC1 silencing was a survival signal, and blockage of autophagy pathway sensitized cancer cells to apoptosis. Finally, we demonstrated that ROC1 was overexpressed in hepatocellular carcinomas, which is associated with poor prognosis of liver cancer patients. These findings suggest that ROC1 is an appealing drug target for liver cancer and provide a proof-of-concept evidence for a novel drug combination of ROC1 inhibitor and an autophagy inhibitor for effective treatment of liver cancer by enhancing apoptosis.

The novel protective role of P27 in MLN4924-treated gastric cancer cells.

The tumor-suppressor gene cyclin-dependent kinase inhibitor 1B (P27) is downregulated in gastric cancer cells mainly through proteolytic degradation mediated by the SKP-Cullin1-F-Box (SCF) complex. But the correlation between its downregulation and gastric cancer prognosis still remains indefinite. MLN4924, an anti-tumor agent, which suppresses the SCF complex by inhibiting Cullin1 neddylation, emerges as a promising tool to elucidate its functions in gastric cancer cells. In this study, MLN4924 induced significant growth inhibition of gastric cancer cells in a dose-dependent manner, along with the simultaneous accumulation of P27 and cell cycle abnormalities such as G2/M arrest. Importantly, we found that P27 silencing in MLN4924-treated cells resulted in an enhancement of growth inhibition both in vitro and in vivo. Mechanism analysis revealed the antagonism effects of antioxidants to this excess apoptosis, suggesting reactive oxygen species (ROS) overproduction especially in the mitochondria was the principal cause of the augmentation. Moreover, the robust ROS attacked the mitochondria to initiate collapse of the mitochondrial membrane permeability and the exportation of apoptosis-inducing factor (AIF), IAP-binding mitochondrial protein (SMAC/DIABLO) and cytochrome c. Finally, we also found that P27 knockdown affected the expression profile of several critical BH3 family members to amplify the mitochondrial dysfunction and apoptosis. In summary, our findings unveiled a protective role of P27 by maintaining mitochondrial membrane permeability in MLN4924-treated gastric cancer cells, and therefore highlighted the potential combination of MLN4924 with P27 inhibition to improve its therapeutic efficacy.

Protective autophagy induced by RBX1/ROC1 knockdown or CRL inactivation via modulating the DEPTOR-MTOR axis.

RBX1/ROC1 is an essential subunit of the largest multiunit Cullin-RING E3 ligase (CRL), which controls the degradation of diverse substrates, thereby regulating numerous cellular processes. Recently, we reported that RBX1 is overexpressed in hepatocellular carcinomas (HCC) and its expression is negatively correlated with patient survival. Moreover, siRNA silencing of RBX1 inhibits the proliferation of liver cancer cells both in vitro and in vivo by inducing CDKN1A/p21-dependent cell senescence. Interestingly, independent of senescence, RBX1 knockdown also triggers an autophagy response, due, at least in part, to the accumulation of the MTOR-inhibitory protein DEPTOR, a recently identified CRL substrate. Biologically, blockage of autophagy significantly enhances the growth-suppressive effect of RBX1 knockdown by triggering massive apoptosis, indicating that the autophagy response upon RBX1 knockdown serves as a survival signal in liver cells. Similar observations were also made in many types of human cancer cells upon inhibition of CRL by MLN4924. These findings suggest that RBX1-CRL is a promising anti-cancer drug target and provide proof-of-concept evidence for a novel drug combination of RBX1-CRL inhibitor and autophagy inhibitor for effective treatment of human cancer.

The down-regulation of GNAO1 and its promoting role in hepatocellular carcinoma

GNAO1 (guanine nucleotide-binding protein, α-activating activity polypeptide O) is a member of the subunit family of Gα proteins, which are molecular switchers controlling signal transductions and whose deregulation can promote oncogenesis. HCC (hepatocellular carcinoma) is one of the malignant tumours around the world, which summons novel biomarkers or targets for effective diagnosis and treatments. The present study was aimed to investigate the expression of GNAO1 in HCC patient tissues and the possible mechanisms by which it took effects. The expression of GNAO1 was detected by IHC (immunohistochemistry) and real-time qPCR (quantitative PCR). Cell proliferation test and cell senescence test were then performed to explore the role of GNAO1 in the occurrence and development of HCC. It was revealed that the level of GNAO1 was comparably less in HCC tissues than in the adjacent tissues. Furthermore, down-regulation of GNAO1 increased cell proliferation, while suppressing the senescence of HCC cells. In conclusion, our findings revealed and confirmed the importance of GNAO1 in HCC, indicating that GNAO1 is a potential biomarker as well as a promising therapeutic target for HCC.

Association between microRNA-499 polymorphism and gastric cancer risk in Chinese population

Single-nucleotide polymorphisms in microRNAs are related to the occurrence, development and prognosis of cancer. The aim of the present study is to investigate the possible influence of the miR-499(rs3746444) polymorphism on the risk of gastric cancer in Chinese population. A total of 363 GC patients and 969 cancer-free controls were enrolled in this study. The genotypes were obtained using MassARRAY method. The results showed that, compared with T allele, C allele was associated with a significantly increased risk of GC (OR=1.491, 95% CI=1.155-1.923, P=0.002). Moreover, a significantly increased risk of GC in subjects with the TC genotype was observed (adjusted OR of 1.559, 95% CI=1.148-2.117, P=0.004), compared with the wide type TT. We also found that basically dominant model (TT vs. TC+CC) was suitable for the association between rs6513497 and the risk of GC (OR=1.568, 95% CI=1.173-2.097, P=0.002). However, the same association was also shown in males and females. Meanwhile, rs3746444 was associated with the tumor size of GC patients. The present study indicated that miR-499 rs3746444 might contribute to GC risk and this SNP could be developed as a biomarker for GC prediction.

Adenosine induces apoptosis through TNFR1/RIPK1/P38 axis in colon cancer cells.

Adenosine, a metabolite of ATP, ubiquitously exists in a wide range of organs and tissues. We previously reported that adenosine was implicated in apoptosis in many cancer cells by extrinsic and/or intrinsic pathways. Here, we found that adenosine suppresses the cell growth by induction of apoptosis of human colonic cancer cells through a novel mechanism. Adenosine suppresses the cell growth of human SW620 and SW480 colon cells in an adenosine transporter and adenosine kinase dependent manner. Moreover, the cell growth suppression is induced by apoptosis through activation of caspase-3 and PARP, and accumulation of ROS in cells. Importantly, we found that adenosine increases the expression of TNFR1 and RIPK1 and the phosphorylation of p38. Knockdown of TNFR1 or RIPK1 impairs the activation of p38, blocks the cleavage of PARP, and provides partially, yet significantly protection from cell death, including reducing the ROS generation in the colon cancer cells. These results indicate that a TNFR1/RIPK1/P38 axis is present in adenosine-induced apoptosis of colonic cancer cells. This axis triggers apoptosis and plays crucial roles in relay of the death signaling. Our study also provides additional experimental evidence for adenosine as a potent therapeutic drug in cancer therapy.

A single nucleotide variant in microRNA-1269a promotes the occurrence and process of hepatocellular carcinoma by targeting to oncogenes SPATS2L and LRP6.

Hepatocellular carcinoma (HCC) is one of the malignant and lethal cancers. Single nucleotide polymorphisms (SNPs) in microRNAs(miRNAs) can affect the expression and target identification of miRNAs and lead to the formation of malignant tumors. However, little is known about whether microRNA-1269a (miR-1269a) SNPs affect the susceptibility and progression of HCC or their specific mechanism. The association between microRNA-1269a rs73239138 and the susceptibility to HCC was verified by MassARRAY assay in a large case-control sample. The effect of miR-1269a and the variant on the proliferation and apoptosis of HCC cells was examined by flow cytometry (FCM), CCK8 assay and Western blot. The target of miR-1269a was identified by bioinformatics analysis and qRT-PCR and its role on cell proliferative capacity was examined by CCK8 assay. The expression level of miR-1269a was analyzed by qRT-PCR in HCC cells transfected with wild or variant type pre-miR-1269a plasmid.MiR-1269a produced a tumor suppressor effect by inhibiting cell proliferation and inducing apoptosis of human HCC cells, possibly via inhibiting the expression of its target genes SPATS2L and LRP6, which were tumor promoters. While, rs73239138 (G>A) in miR-1269a reduced the anticancer effect of miR-1269a possibly by attenuating its total amount in HCC cells or its target recognition, reduce its inhibition on target genes and promoted the susceptibility to HCC. Our findings for the first time proved that miR-1269a SNP plays a role in the occurrence and process of HCC and the relevant mechanism, in accompany with the discovery of the novel target genes of miR-1269a.

Screening and verification of ssDNA aptamers targeting human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most malignant cancers in the world. Molecular probes that can recognize biomarkers specific for HCC are urgently needed to improve the sensitivity and specificity of early diagnosis. A recent study has applied the method of systematic evolution of ligands by exponential enrichment and produced several aptamers that can bind specifically to mouse liver cancer cells and tissues. However, the binding affinity to human liver cancer has not been fully identified. Using human-derived hepatoma cell line HepG2 as positive target cell line and normal hepatocyte cell line HL-7702 as negative one, we obtained an aptamer HA09 that could specifically bind to human liver cancer cells with Kd in the nanomolar range and recognize paraffin-embedded human HCC tissues. This aptamer may facilitate the discovery of novel biomarkers and serve as an ideal molecular probe for intracellular delivery with both diagnostic and therapeutic implications.

Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer.

Bax is a key molecule in mitochondria-apoptosis pathway, however it is not always an efficient apoptosis inducer in chemotherapeutic agents-treated cancer cells. Here, we found that specific inhibition of AURKA by MLN8237-induced calpain-mediated Bax cleavage at N-terminal 33th asparagine (c-Bax) to promote apoptosis. The c-Bax, as Bax, could also efficiently located to mitochondria but c-Bax is a stronger apoptosis inducer than Bax. Morever, c-Bax-induced apoptosis could not be blocked by the canonical Bax inhibitor, Bcl-2. Further study found p27 was degraded and subsequently Bax was transformed to c-Bax through calpain. Also, p27 efficiently inhibited Bax cleavage and p27 knockdown sensitized apoptosis through Bax cleavage when cancer cells were treated with MLN8237. It is also demonstrated that the anti-apoptotic role of p27 lies its cytoplasmic localization. Finally, we found that the positive correlation between AURKA and p27 in advanced gastric cancer patients. In conclusion, we found that MNL8237 suppressed cell growth by regulating calpain-dependent Bax cleavage and p27 dysregulation in gastric cancer cells.

Association between microRNA single nucleotide polymorphisms and the risk of hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma (HCC) has a high morbidity and mortality. Single nucleotide polymorphisms (SNPs) of microRNA (miRNA) may be associated with the susceptibility to develop certain malignant tumors. AIM To study the association between SNPs of miRNA and hepatocellular carcinoma in peripheral blood samples. MATERIAL AND METHODS Three SNPs in miRNA were studied in peripheral blood samples of 498 patients with HCC and 520 controls. RESULTS A significant association was observed between rs13299349 in miRNA3152 and HCC. AA genotype or A allele were significantly associated with increased risk of HCC. A allele was associated with the size and number of tumor foci. There was also a relationship between rs10061133 in miRNA449b and HCC. The G allele was significantly associated with increased risk of HCC compared with A allele. CONCLUSIONS This study links rs13299349 in miRNA3152 and rs10061133 in miRNA449b with the risk of developing HCC.