A five-pseudouridylation-associated-LncRNA classifier for primary prostate cancer prognosis prediction.

Background: Prostate cancer (PCa) is one of the most common cancers in males around the globe, and about one-third of patients with localized PCa will experience biochemical recurrence (BCR) after radical prostatectomy or radiation therapy. Reportedly, a proportion of patients with BCR had a poor prognosis. Cumulative studies have shown that RNA modifications participate in the cancer-related transcriptome, but the role of pseudouridylation occurring in lncRNAs in PCa remains opaque. Methods: Spearman correlation analysis and univariate Cox regression were utilized to determine pseudouridylation-related lncRNAs with prognostic value in PCa. Prognostic pseudouridylation-related lncRNAs were included in the LASSO (least absolute shrinkage and selection operator) regression algorithm to develop a predictive model. KM (Kaplan-Meier) survival analysis and ROC (receiver operating characteristic) curves were applied to validate the constructed model. A battery of biological cell assays was conducted to confirm the cancer-promoting effects of RP11-468E2.5 in the model. Results: A classifier containing five pseudouridine-related lncRNAs was developed to stratify PCa patients on BCR and named the "ψ-lnc score." KM survival analysis showed patients in the high ψ-lnc score group experienced BCR more than those in the low ψ-lnc score group. ROC curves demonstrated that ψ-lnc score outperformed other clinical indicators in BCR prediction. An external dataset, GSE54460, was utilized to validate the predictive model's efficacy and authenticity. A ceRNA (competitive endogenous RNA) network was constructed to explore the model's potential molecular functions and was annotated through GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses. RP11-468E2.5 was picked for further investigation, including pan-cancer analysis and experimental validation. Preliminarily, RP11-468E2.5 was confirmed as a tumor promoter. Conclusion: We provide some evidence that pseudouridylation in lncRNA played a role in the development of PCa and propose a novel prognostic classifier for clinical practice.

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.

SOX2 regulates multiple malignant processes of breast cancer development through the SOX2/miR-181a-5p, miR-30e-5p/TUSC3 axis.

BACKGROUND: High levels of SOX2 protein are correlated with increased dissemination of breast cancer. However, the underlying molecular mechanisms are not fully understood. METHODS: In this study we investigate the role of SOX2 in breast cancer metastasis using multiple in vitro and in vivo assays including cell culture, shRNA-mediated knockdown, wound healing, colony formation, transwell chamber, xenograft and tail vein injection. Moreover, western blot, immunostaining, microarray and real-time PCR were used to determine the change of protein and miRNA levels. Luciferase assays were also used to evaluate activity which TUSC3 is a target of miR-181a-5p and miR-30e-5p, and the clinical survival relevance was analyzed by Kaplan-Meier analysis. RESULTS: We identified a novel pathway involving SOX2 regulation of microRNAs to control the proliferation and migration of breast cancer cells. shRNA-mediated knockdown of SOX2 inhibits breast cancer cell expansion and migration. More importantly, we found that these changes are accompanied by significant reduction in the levels of two microRNAs, miR-181a-5p and miR-30e-5p. Overexpression of these two microRNAs leads to reduced protein levels of Tumor Suppressor Candidate 3 (TUSC3) in breast cancer cells; mutations of the potential binding sites in the 3'-UTR of TUSC3 abrogate the inhibitory effects of the microRNAs. We further found that upregulation of TUSC3 expression leads to reduced proliferation and migration of breast cancer cells. In human breast cancer samples the levels of TUSC3 protein are inversely correlated with those of SOX2 protein. CONCLUSIONS: Taken together, our work reveals a novel SOX2-mediated regulatory axis that plays critical roles in the proliferation, migration and invasiveness of breast cancer cells. Targeting this axis may provide beneficial effect in the treatment of breast cancer.

Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A.

As an immunotoxin, diphtheria toxin has been widely used in gene therapy and gene function assays for its roles in protein synthesis inhibition, and the aim of our study is to set up a nonintegrating lentiviral system for specific expression of diphtheria toxin A (DTA) used in cancer gene therapy.Here, we established a lentiviral system that could coordinately express fluorescent protein and DTA driven by the cytomegalovirus (CMV) promoter, which is convenient for us to precisely trace the expression of DTA and monitor the process of lentivirus packaging. To achieve safer cancer therapy, we replaced the CMV promoter with the Survivin promoter, a specific promoter that is dramatically activated in cancer tissues and cells, but not in normal tissues and cells, and that will impose greater therapeutic potential because a significant expression difference occurred between these 2 groups. Meanwhile, we obtained integrase-deficient lentivirus (IDLV) after packaging with the integrase mutant, which expresses defective integrase RRK262263264AAH, to minimize the side effects that derived from the insertional mutagenesis of the host genome.Our results suggest that the IDLV system that we generated possesses therapeutic potential in cancers in vitro and in vivo.

Integrase-deficient lentivirus: opportunities and challenges for human gene therapy.

Lentiviruses are powerful tools for gene delivery and have been widely used for the dissection of gene functions in both replicating and quiescent cells. Recently, lentiviruses have also been used for delivering target sequences in gene therapy. Although the lentiviral system provides sustained exogenous gene expression, serious concerns have been raised due to its unfavorable insertion-mediated mutagenesis effect, thereby resulting in the silencing or activation of some unexpected genes. Thus, an array of modifications of the original vectors may reduce risks. Here, we briefly review the structure of the integrase protein, which is an essential protein for viral insertion and integration; the mechanisms of integrase-mediated integration; and the effects of the modifications of integrase. Moreover, we discuss the advantages resulting from integrase modifications and their future applications. Taken together, the generation of integrase-deficient lentivirus (IDLV) not only provides us with an opportunity to reduce the risk of virus-mediated insertions, which would improve the safety of gene therapy, but also favors gene correction and vaccine development.

The multiple roles for Sox2 in stem cell maintenance and tumorigenesis.

The Sry-containing protein Sox2 initially was known to regulate the self-renewal of the mouse and human embryonic stem cells (ESCs). It is also important for the maintenance of stem cells in multiple adult tissues including the brain and trachea, and it is one of the key transcription factors for establishing induced pluripotent stem cells. Recently, overexpression and gene amplification of Sox2 have been associated with the development of squamous cell carcinoma in multiple tissues such as the lung and esophagus. These different roles for Sox2 involve a complicated regulatory networks consisting of microRNAs, kinases and signaling molecules. While the levels of Sox2 are modulated transcriptionally and translationally, post-translational modification is also important for the various functions of Sox2. In clinics, high levels of Sox2 are correlated with poor prognosis and increased proliferation of cancer stem cells. Therefore targeting Sox2 can be potentially explored for a new therapeutic avenue to treat cancers. This review will focus on the different roles for Sox2 in stem cell maintenance and its oncogenic roles in the context of signal transcription and microRNA regulation. We will also review the main upstream and downstream targets of Sox2, which can be potentially used as therapeutic measures to treat cancer with abnormal levels of Sox2.