RNA-Binding Protein La Mediates TGFβ-Induced Epithelial to Mesenchymal Transition and Cancer Stem Cell Properties

Background: the aberrant overexpression of predominantly nuclear localizing RNA-binding protein (RBP) La contributes to proliferation, mobility, and chemoresistance of cancer cells and tumor growth in mice. Methods: studies included cancer tissue microarrays (TMAs) analyses, cancer tissue data mining, transforming growth factor β (TGFβ)-induced cancer cell plasticity studies, three dimensional sphere growth, epithelial to mesenchymal transition (EMT) assays, analysis of cancer stem cell (CSC) marker expression, and post-translational modification of cancer-associated La protein. Results: we demonstrated that significant overexpression of RBP La in lung and head and neck cancer tissue correlates with poor overall survival. Furthermore, small interfering RNA-mediated depletion of La reduced proliferation and migration of cancer cells, blocked TGFβ-induced EMT, and diminished both EMT and CSC marker expression. Rescue experiments with La wildtype but not RNA chaperone domain activity-defective La mutant increased the expression of those cancer progression markers, suggesting a critical role of La’s RNA chaperone activity in this process. La depletion in cancer cells also significantly decreased sphere growth in the presence of TGFβ. Interestingly, TGFβ treatment induced phosphorylation of La at threonine 389 (pLaT389) only in adherents but not in 3D growing cultures. Conclusion: our study suggests that the TGFβ/AKT/pLaT389 signaling pathway regulates cancer cell plasticity

The hepatitis B virus PRE contains a splicing regulatory element

The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein–protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE

Evaluating the RIST Molecular-Targeted Regimen in a Three-Dimensional Neuroblastoma Spheroid Cell Culture Model

Abstract Background: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as ‘pre-treatment’ with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. Methods: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients’ data mining. Results: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted ‘pre-treatment’ of spheroids decreased neoplastic signaling and CSC marker expression. Conclusions: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties

Irinotecan and temozolomide in combination with dasatinib and rapamycin versus irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma (RIST-rNB-2011): a multicentre, open-label, randomised, controlled, phase 2 trial

Background Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan–temozolomide and dasatinib–rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. Methods The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1–25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan–temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2–4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin–dasatinib and irinotecan–temozolomide for four cycles over 8 weeks, then two courses of rapamycin–dasatinib followed by one course of irinotecan–temozolomide for 12 weeks) with irinotecan–temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. Findings Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7–8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31–88), the median progression-free survival was 11 months (95% CI 7–17) in the RIST group and 5 months (2–8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4–24) in the RIST group versus 2 months (2–5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9–7) in the RIST group versus 8 months (4–15) in the control group (HR 0·84 [95% CI 0·51–1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). Interpretation RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting

Role of the RNA-binding protein La in cancer pathobiology

RNA-binding proteins are important regulators of RNA metabolism and are of critical importance in all steps of the gene expression cascade. The role of aberrantly expressed RBPs in human disease is an exciting research field and the potential application of RBPs as a therapeutic target or a diagnostic marker represents a fast-growing area of research. Aberrant overexpression of the human RNA-binding protein La has been found in various cancer entities including lung, cervical, head and neck, and chronic myelogenous leukaemia. Cancer-associated La protein supports tumour-promoting processes such as proliferation, mobility, invasiveness and tumour growth. Moreover, the La protein maintains the survival of cancer cells by supporting an anti-apoptotic state that may cause resistance to chemotherapeutic therapy. The human La protein represents a multifunctional post-translationally modified RNA-binding protein with RNA chaperone activity that promotes processing of non-coding precursor RNAs but also stimulates the translation of selective messenger RNAs encoding tumour-promoting and anti-apoptotic factors. In our model, La facilitates the expression of those factors and helps cancer cells to cope with cellular stress. In contrast to oncogenes, able to initiate tumorigenesis, we postulate that the aberrantly elevated expression of the human La protein contributes to the non-oncogenic addiction of cancer cells. In this review, we summarize the current understanding about the implications of the RNA-binding protein La in cancer progression and therapeutic resistance. The concept of exploiting the RBP La as a cancer drug target will be discussed

Heterogeneity and Common Features of Defective Hepatitis B Virus Genomes Derived from Spliced Pregenomic RNA

AbstractDefective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional “spliced” genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis

The RNA chaperon activity of the human La protein (LARP3): DOI: 10.14800/rd.872

Single-stranded RNA molecules fold intensively into secondary and tertiary structures and are often trapped in non-functional configurations. To adapt a functional configuration, structural changes have to be achieved. RNA helicases and RNA chaperones are proteins able to assist those structural rearrangements in an ATP-dependent or ATP-independent manner, respectively. The cancer-associated RNA-binding protein La (LARP3) is an RNA chaperone involved in various aspects of the RNA metabolism. Recently the RNA chaperone domain within the human La protein has been mapped and demonstrated that its activity is required to stimulate cyclin D1-internal ribosome entry site (IRES)-dependent protein synthesis. Furthermore, it has been shown that the La protein can be phosphorylated by serine/threonine kinase AKT in vitro. Taken together, we suggest a model in which the RNA chaperone La stimulates translation of specific target mRNAs by assisting structural changes in their translation start site surrounding RNA region

Comparing mTOR inhibitor Rapamycin with Torin-2 within the RIST molecular-targeted regimen in neuroblastoma cells

The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC50 values of Torin-1, Torin-2, AZD3147 and PP242 we established that only Torin-2 inhibited cell viability of all three MycN-amplified neuroblastoma cell lines tested at nanomolar concentration. Single treatment of both mTOR inhibitors induced a significant G1 cell cycle arrest and combination treatment with Dasatinib reduced the expression of cell cycle regulator cyclin D1 or increased the expression of cell cycle inhibitor p21. The combinatorial index depicted for both mTOR inhibitors a synergistic effect with Dasatinib. Interestingly, compared to Rapamycin, the combination treatment with Torin-2 resulted in a broader mTOR pathway inhibition as indicated by reduced phosphorylation of AKT (Thr308, Ser473), 4E-BP (Ser65), and S6K (Thr389). Furthermore, substituting Rapamycin in the modified multimodal RIST protocol with Torin-2 reduced cell viability and induced apoptosis despite a significant lower Torin-2 drug concentration applied. The efficacy of nanomolar concentrations may significantly reduce unwanted immunosuppression associated with Rapamycin. However, at this point we cannot rule out that Torin-2 has increased toxicity due to its potency in more complex systems. Nonetheless, our results suggest that including Torin-2 as a substitute for Rapamycin in the RIST protocol may represent a valid option to be evaluated in prospective clinical trials for relapsed or treatment-refractory high-risk neuroblastoma