RNF4 silencing induces cell growth arrest and DNA damage by promoting nuclear targeting of p62 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the largest causes of cancer-related deaths worldwide owing to the limitation of effective treatment options. The ubiquitin-proteasome system has been rapidly recognized as a frequent target of deregulation leading to cancers. Enhanced DNA damage response (DDR) promotes HCC growth and prevents chemosensitivity, and ubiquitin E3 ligases are key modulators in DDR. Therefore, a better understanding of how E3 ligases regulate cell growth and DNA damage may provide novel insights in understanding the oncogenic mechanism and improving the efficacy of DNA damage therapeutic agents. Here, we performed a high-content RNAi screening targeting 52 DDR-related E3 ligases in HCC and found that ring finger protein 4 (RNF4) was essential for HCC growth. RNF4 was highly expressed in HCC tissues, and the expression levels of RNF4 were associated with poor outcomes. RNF4 silencing significantly suppressed the cell growth, and subsequently induced G2/M arrest and apoptosis of HCC cells in vitro; RNF4 silencing also demonstrated the tumor-suppressive efficacy on HCC in vivo. Moreover, RNF4 silencing increased DNA damage, and rendered HCC cells more sensitive to DNA damage drugs and radiation. We found RNF4 functionally interacts with p62, and mechanistic analyses indicated that RNF4 silencing triggered the nuclear enrichment of p62. Moreover, the p62 nuclear targeting was required for increased DNA damage and growth suppression mediated by RNF4 silencing. Thus, our findings suggest RNF4 is essential for HCC proliferation via preventing nuclear translocation of p62. RNF4 silencing promotes DNA damage and may serve as a novel strategy to suppress cell growth and increase the sensitivity of DNA damage therapeutic agents in HCC.

SINO Syndrome Causative KIDINS220/ARMS Gene Regulates Adipocyte Differentiation.

Nonsense variants in KIDINS220/ARMS were identified as the main cause of spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO) syndrome, a rare disease with birth defects in brachycephaly, neurological disorder, and obesity. The cause of neural cell dysfunction by KIDINS220/ARMS were extensively studied while the cause of obesity in SINO syndrome remains elusive. Here, we identified KIDINS220/ARMS as an adipocyte differentiation-regulating gene. A Chinese family, mother and her two sons, all showed severe symptoms of SINO syndrome. G-banding karyotyping, chromosome microarray analysis, and whole exome sequencing revealed a novel amber mutation, c.3934G>T (p. E1312X), which was close to the C-terminal region of KIDINS220/ARMS and resulted in the premature of the protein. Both the mRNA and protein levels of KIDINS220/ARMS gradually decreased during adipocyte differentiation. Knockdown of KINDINS220/ARMS could prompt adipocyte differentiation and lipid accumulation while overexpression of KIDINS220/ARMS decrease the rate of matured adipocytes. Furthermore, we demonstrated that KIDINS220/ARMS inhibits adipocyte maturation through sustained extracellular signal-regulated kinase signaling. In conclusion, this is the first report about a vertical heredity of severe dominant pathogenic mutation of KIDINS220/ARMS, suggested that KIDINS220/ARMS played a negative role in adipocyte maturation, explained the cause of obesity in SINO syndrome and could highlight the importance of adipocyte differentiation in neuron functions.

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.

Targeting of the C-Jun/BCL-XL/P21 Axis Accelerates the Switch from Senescence to Apoptosis Upon ROC1 Knockdown in Gastric Cancer Cells.

BACKGROUND/AIMS: Regulator of cullins-1 (ROC1) is a pivotal component of cullin-RING E3 ubiquitin ligases, which help to regulate distinct cellular processes by governing the degradation of various substrates. Because the role of ROC1 in gastric cancer is largely uncharacterized, we investigated the relationship between ROC1 expression and the prognosis of gastric cancer patients and explored the biological function of ROC1 and its underlying mechanisms in gastric cancer. METHODS: Kaplan-Meier and multivariate Cox regression analyses were used to evaluate the correlation between the carcinogenesis of gastric cancer and ROC1. SA-ß-gal staining and the senescence-associated secretory phenotype (SASP) were used to assess ROC1 silencing-induced cellular senescence. A xenograft zebrafish model was used to evaluate the effects of BCL-XL and ROC1 co-silencing on tumor formation in vivo. RESULTS: High ROC1 expression was correlated with poor prognosis and a low 5-year survival rate of gastric cancer patients. ROC1 depletion significantly inhibited the growth of gastric cancer cells by sequentially inducing p21-mediated cellular senescence and mitochondrial-dependent apoptosis. Functional studies revealed successive upregulation of c-Jun, BCL-XL, and p21 upon ROC1 knockdown. BCL-XL suppression via RNA interference or a BH3 mimetic (ABT-737 or ABT-263) efficiently enhanced the anti-tumor effects of ROC1 knockdown. Equally as important, BCL-XL silencing strengthened ROC1 knockdown-induced apoptosis by blocking p21-mediated senescence. CONCLUSIONS: The c-Jun/BCL-XL/p21 axis promotes senescence to resist ROC1 knockdown-induced apoptosis in gastric cancer cells. Targeted inactivation of BCL-XL could sensitize gastric cancer cells to ROC1 knockdown in clinical practice.

Targeting CAND1 promotes caspase-8/RIP1-dependent apoptosis in liver cancer cells.

Cullin-associated NEDD8-dissociated 1 (CAND1) plays a vital role in regulating the activity of Cullin-RING ubiquitin ligases (CRLs), which are frequently dysregulated in cancer. However, the role of CAND1 in hepatocellular carcinoma (HCC) remains unknown. Here, we found that CAND1 was overexpressed in HCC tissues compared to corresponding adjacent liver tissues (71.7% vs 16.7%); high expression of CAND1 was associated with poor overall survival (40.7 vs 57.3 months, P=0.0013); and CAND1 was an independent risk factor for the prognosis of HCC patients (N=138, P=0.018). Functional studies revealed that CAND1 knockdown efficiently suppressed the proliferation of liver cancer cells by activating caspase-8-dependent mitochondrial apoptosis. We also observed a mutual activation loop between caspase-8 and Receptor Interacting Protein 1 (RIP1), which amplified CAND1 knockdown-induced apoptotic signals in the cells. Furthermore, RIP1 inhibitor Necrostatin-1 eliminated the activation of caspase-8. In conclusion, our study pioneered in reporting high CAND1 expression as a predictor of poor prognosis for HCC patients. CAND1 silencing suppressed HCC cell proliferation by inducing caspase-8/RIP1-dependent apoptosis. These findings supported that CAND1 could be a new therapeutic target for liver cancer.

Targeting high Aurora kinases expression as an innovative therapy for hepatocellular carcinoma.

The Aurora kinases A and B control tumorigenesis by inhibiting apoptosis and promoting proliferation and metastasis, however, it remains unknown whether Aurora A and B overexpressed concomitantly and its clinical significance in hepatocellular carcinoma (HCC). Here, we obsearved Aurora A and B tended to overexpress parallelly on protein level (r = 0.8679, P < 0.0001) and their co-overexpression (Aurora AHBH), associated with the worst prognosis, was an independent predictor for the survival. Importantly, with the lower IC50 and stronger anti-tumor effect than selective inhibitors, SNS-314, the pan-inhibitor of Aurora kinases, which induced YAP (Yes-associated protein) reduction and resulted in P21 accumulation, significantly promoted the polyploidy (> 4N) formation and apoptosis in HCC. High YAP expression (YAPH) was associated with Aurora AHBH, and appeared to be an independent predictor for survival, but P21 not. Moreover, silencing YAP also induced P21 accumulation, and knockdown P21, which enhanced YAP accumulation and weakened the SNS-314-induced YAP reduction, impaired SNS-314-induced apoptosis. Therefore, P21 enhanced the apoptotic effect of SNS-314 in HCC. Taken together, our findings indicated Aurora kinases/YAP/P21 was an oncogenic signaling axis in HCC, and revealed targeting Aurora AHBH induced apoptosis by YAP suppression. Our results also provided a solid evidence for SNS-314 as a potential targeted therapy, and a proof-of-concept evidence for a possible combined therapy of SNS-314 plus Hippo pathway inhibitors on HCC.

Netrin-4 as a biomarker promotes cell proliferation and invasion in gastric cancer.

Gastric cancer (GC) is the second most common cause of cancer-related death with limited serum biomarkers for diagnosis and prognosis. Netrin-4 (Ntn4) is a laminin-related secreted molecule found to regulate tumor progression and metastasis. However, it is completely unknown whether Ntn4 has roles in GC development. Here, we first reported Ntn4 knockdown significantly suppressed cell proliferation and motility, while overexpression or addition of exogenous Ntn4 reversed these effects. In addition, Ntn4 receptor, neogenin (Neo) was also found highly expressed in GC cells and mediated the Ntn4-induced cell proliferation and invasion. Moreover, Ntn4 or Neo silencing decreased the phosphorylation of Stat3, ERK, Akt and p38, indicating multi-oncogenic pathways (Jak/Stat, PI3K/Akt, and ERK/MAPK) were involved in Ntn4-induced effects on the GC cells. Importantly, Ntn4 level was significantly increased in 82 tumor tissues (p = 0.001) and 52 serum samples (p < 0.0001) from GC patients and positively correlated with Neo expression (p = 0.003). Ntn4 expression was negatively correlated with the survival period (p = 0.038), and positively associated with the severity of pathological stages of the tumors (p = 0.008). Taken together, Ntn4 promoted the proliferation and motility of GC cells which was mediated by its receptor Neo and through further activation of multi-oncogenic pathways. Elevated Ntn4 was detected in both tumor tissues and serum samples of GC patients and suggested a relatively poor survival, indicating Ntn4 may be used as a potential non-invasive biomarker for diagnosis and prognosis of GC.

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.