Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway

The neural ELAVL protein HuB enhances endogenous proto-oncogene activation

The cytoplasmic distribution of the HuR/ELAVL1 (embryonic lethal abnormal vision 1) protein is recognized as an important prognostic factor of malignant tumors. However, the previous study suggests that exogenous over-expression of HuR is not sufficient for nuclear export. Conversely, the predominantly cytosolic distribution of neuron-specific human ELAV members, including HuB/ELAVL2, HuC/ELAVL3, and HuD/ELAVL4, has been reported. In the present study, we demonstrated the expression of HuB in several types of cancer cells, but expression of HuC and HuD was not observed. In addition, our results indicated that HuR and HuB formed a complex in the cytosolic fraction of cancer cells via the RRM3 region. Ectopic expression of HuB was capable of initiating the cytosolic translocation of HuR from the nucleus to the cytosol. Furthermore, HuB-transduced cancer cells displayed significant nuclear export of HuR, with quantitative PCR experiments revealing the simultaneous upregulation of HIF-1 alpha, c-Fos, c-MYC, and Ets2 basal mRNA expression. Phorbol 12-myristate 13-acetate (PMA)-stimulated HuB-transduced cells demonstrated significantly enhanced activation of endogenous c-Fos and CREB dependent cascades. Finally, co-transfection of HuB with the El region of type 5 human adenovirus significantly enhanced El transformation activities but that of HuR with the El region did not. Collectively, our findings suggest that the neural Hu family protein HuB plays a major role in the activation of memory-related proto-oncogenes. (C) 2019 Elsevier Inc. All rights reserved

HuR Knockdown Changes the Oncogenic Potential of Oral Cancer Cells

HuR binds to AU-rich element (ARE) containing mRNA to protect them from rapid degradation. Here, we show that knockdown of HuR changes the oncogenic properties of oral cancer cells. Oral squamous cell carcinoma cell lines, HSC-3 and Ca9.22, which express HuR protein and cytoplasmic ARE-mRNA more abundantly than normal cells, were subjected to HuR knockdown. In the HuR-knockdown cancer cells, the cytoplasmic expression of c-fos, c-myc, and COX-2 mRNAs was inhibited compared to those in cells that had been transfected with a control siRNA, and the half-lives of these mRNAs were shorter than those of their counterparts in the control cells. HuR-knockdown cells failed to make colonies in soft agar, suggesting that the cells had lost their ability for anchorage-independent cell growth. Additionally, the motile and invasive activities of the cells decreased remarkably by HuR knockdown. Furthermore, the expression of cell cycle-related proteins, such as cyclin A, cyclin B1, cyclin D1, and CDK1, was reduced in the HuR-knockdown cancer cells, and HuR bound to cdk1 mRNA in order to stabilize it. These findings suggest that HuR knockdown changes the features of oral cancer cells, at least in part, by affecting their cell cycle, and shows potential as an effective therapeutic approach

Human adenovirus oncolytic properties and the inhibitory role of E4 orf4 and E4 orf6/7 on endogenously activated NF-κB

Human adenovirus is a promising tool for cancer therapy as an oncolytic virus. To predict which region of the oncolytic adenovirus E4 gene could be deleted, we investigated the relationship between the E4 proteins and NF-κB. Here, we report that TLR2-dependent NF-κB activation in Ad5-infected cells was significantly inhibited 24 h post-infection. Among the six E4 proteins, E4 orf4 and E4 orf6/7 exhibited notable suppressive effects on NF-κB activation. However, only E4 orf4 was co-immunoprecipitated with the RelA protein, also known as p65. It appears likely that E4 orf6/7 represses NF-κB activation via E2F-dependent pathways. Our results suggest that both E4 orf4 and E4 orf6/7 are novel inhibitors of NF-κB activation. The inhibition of endogenous NF-κB activation by E4 proteins during the late phase of infection also appears to elucidate the previously reported suppression of E1A expression in the late phase of infection. These redundant suppressive effects of E4 orf4 and E4 orf6/7 on NF-κB suggest that these proteins may play a major role in the anticancer properties of oncolytic adenovirus

pp32r1 controls the decay of the RNA- binding protein HuR

pp32 is a tumor suppressor and is one of the associated proteins of the RNA-binding protein HuR. The pp32-HuR complex is exported to the cytoplasm of cells under stress conditions, and HuR is degraded by caspases in the cytoplasm. In the present study, we examined the role of pp32r1, a member of the pp32 family that has oncogenic properties, in the decay of HuR. pp32r1 was found to be abundantly expressed in cancer cells, and overexpression of pp32r1 induced colony formation in soft-agar. pp32r1 was expressed in both the nucleus and cytoplasm, whereas pp32 was predominantly localized in the nucleus. Even with lethal stress such as staurosporine (STS), HuR in the cytoplasm was never downregulated, and caspase-3 activity was inhibited when cells expressed pp32r1. pp32r1 bound to HuR without interacting with pp32. In cancer cells, HuR survived in the cytoplasm of cells overexpressing pp32r1, although HuR was not expressed in the cytoplasm of pp32-expressing cells, similar to lethal stress conditions. Taken together, these results indicate that pp32r1 binds to HuR to avoid the caspase-mediated decay of HuR in the cytoplasm of cells. We suggest that this function contributes to the oncogenic activity of pp32r1

ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia

Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk