Genome Doubling Shapes High-Grade Transformation and Novel EWSR1::LARP4 Fusion Shows SOX10 Immunostaining in Hyalinizing Clear Cell Carcinoma of Salivary Gland.

Hyalinizing clear cell carcinoma (HCCC) is a rare indolent malignant tumor of minor salivary gland origin with EWSR1::ATF1 rearrangement. Pathologically, the tumor cells possess a clear cytoplasm in a background of hyalinized stroma. Generally, the tumor cells are positive for p63 and p40 and negative for s100 and α-smooth muscle actin, suggesting that they differentiate into squamous epithelium and not into myoepithelium. In this study, we performed a detailed histopathological and genomic analysis of 6 cases of HCCC, including 2 atypical subtypes-a case of "high-grade transformation" and 1 "possessing a novel partner gene for EWSR1." We performed a sequential analysis of the primary and recurrent tumor by whole-exome sequencing, RNA sequencing, Sanger sequencing, and fluorescence in situ hybridization to investigate the effect of genomic changes on histopathology and clinical prognosis. A fusion gene involving the EWSR1 gene was detected in all cases. Five cases, including the "high-grade transformation," harbored a known EWSR1::ATF1 fusion gene; however, 1 case harbored a novel EWSR1::LARP4 fusion gene. This novel EWSR1::LARP4-fused HCCC has a SOX10-positive staining, which is different from the EWSR1::ATF1-fused HCCC. According to whole-exome sequencing and fluorescence in situ hybridization analysis, the "whole-genome doubling" and focal deletion involving CDKN2A, CDKN2B, and PTEN were detected in HCCC with "high-grade transformation." Conclusively, we identified a novel partner gene for EWSR1, LARP4, in indolent HCCC. Importantly, "high-grade transformation" and poor prognosis were caused by whole-genome doubling and subsequent genomic aberrations.

Up-regulation of circ_LARP4 suppresses cell proliferation and migration in ovarian cancer by regulating miR-513b-5p/LARP4 axis.

BACKGROUND: Ovarian cancer (OC) is a common fatal malignant tumor of female reproductive system worldwide. Growing studies have proofed that circular RNAs (circRNAs) engage in the regulation of various types of cancers. However, the underlying biological functions and effect mechanism of circular RNA_LARP4 (circ_LARP4) in OC have not been explored. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to detect the expression of circ_LARP4 in OC cells. The function of circ_LARP4 was measured by cell counting kit-8 (CCK-8), colony formation assay and transwell assay. RNA immunoprecipitation (RIP) assay and luciferase reporter assays assessed the binding correlation between miR-513b-5p and circ_LARP4 (or LARP4). RESULTS: The expression of circ_LARP4 in OC cells was much lower than that in human normal ovarian epithelial cells. Overexpressing circ_LARP4 impaired cell proliferation, invasion and migration abilities. Circ_LARP4 worked as a competing endogenous RNA (ceRNA) to sponge miR-513b-5p. Furthermore, LARP4 was indirectly modulated by circ_LARP4 as the downstream target of miR-513b-5p, as well as the host gene of circ_LARP4. CONCLUSION: Circ_LARP4 could hamper cell proliferation and migration by sponging miR-513b-5p to regulate the expression of LARP4. This research may provide some referential value to OC treatment.

Circular RNA La-related RNA-binding protein 4 correlates with reduced tumor stage, as well as better prognosis, and promotes chemosensitivity to doxorubicin in breast cancer.

OBJECTIVE: We aimed to evaluate the correlation of circular RNA La-related RNA-binding protein 4 (circ-LARP4) with tumor characteristics and prognosis, and its effect on chemosensitivity in breast cancer. METHODS: Circ-LARP4 from tumor and adjacent tissues of 283 female breast cancer patients underwent resection was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor features, disease-free survival (DFS), and overall survival (OS) were recorded. In vitro, circ-LARP4 in human normal mammary epithelial cells (HMEC) and breast cancer cell lines was detected by RT-qPCR. MCF-7 and MDA-MB-231 cells were transfected with circ-LARP4 overexpression plasmid (as OE-Circ group) and control overexpression plasmid (as OE-Control group). Relative cell viability under different concentrations of doxorubicin was measured. RESULTS: Circ-LARP4 was decreased in tumor tissues than adjacent tissues (P < .001). Tumor circ-LARP4 negatively correlated with tumor size (P = .001), T stage (P = .009), N stage (P = .006), and TNM stage (P < .001), whereas positively correlated with DFS (P = .004) and OS (P < .001). In vitro, circ-LARP4 was decreased MCF-7, BT474, MDA-MB-231, and MDA-MB-468 cell lines than HMEC (all P < .001). Relatively cell viability of MCF-7 cells (at 20 nmol/L [P < .05], 40 nmol/L [P < .01], 80 nmol/L [P < .05] of doxorubicin) and MDA-MB-231 cells (at 120 nmol/L [P < .05], 240 nmol/L [P < .05] of doxorubicin) was decreased in OE-Circ group than OE-Control group. IC50 value of doxorubicin was decreased in OE-Circ group than OE-Control group in MCF-7 and MDA-MB-231 cell lines (both P < .01). CONCLUSION: Circ-LARP4 was a potential prognostic biomarker, which might improve the management of breast cancer.

Circular RNA LARP4 correlates with decreased Enneking stage, better histological response, and prolonged survival profiles, and it elevates chemosensitivity to cisplatin and doxorubicin via sponging microRNA-424 in osteosarcoma.

BACKGROUND: This study aimed to evaluate the association of circular RNA La-related RNA-binding protein 4 (circ-LARP4) with clinical features and prognosis in osteosarcoma patients, and further explore its effect on chemosensitivity in osteosarcoma cells. METHODS: Seventy-two osteosarcoma patients with Enneking stage IIA-IIB who underwent resection were consecutively enrolled, and then, tumor tissues and non-tumor tissues were obtained. Circ-LARP4 in tumor tissue/non-tumor tissue was detected by quantitative polymerase chain reaction. After circ-LARP4 overexpression and negative control overexpression plasmid transfection, relative cell viability (%) was evaluated by Cell Counting Kit-8 in MG63 cells treated by different concentrations of cisplatin, methotrexate, and doxorubicin, and IC50 was calculated. RESULTS: Circ-LARP4 was downregulated in tumor tissue compared with non-tumor tissue and had a good value in distinguishing tumor tissue from non-tumor tissue with an area under curve of 0.829 (95% CI: 0.762-0.859). Meanwhile, tumor circ-LARP4 was negatively correlated with the Enneking stage. After resection, circ-LARP4 high expression patients showed an increased tumor cell necrosis rate to adjuvant chemotherapy compared to circ-LARP4 low expression patients, and circ-LARP4 high expression correlated with prolonged disease-free survival and overall survival. In vitro experiments revealed that circ-LARP4 overexpression elevated the chemosensitivity of MG63 cells to cisplatin and doxorubicin but not methotrexate, with decreased cisplatin IC50 and doxorubicin IC50 concentrations than negative control. Besides, miR-424 overexpression attenuated the chemosensitivity in circ-LARP4 overexpression-treated MG63 cells. CONCLUSION: Circ-LARP4 high expression correlates with decreased Enneking stage and prolonged survival profiles, and it elevates chemosensitivity to cisplatin and doxorubicin via sponging miR-424 in osteosarcoma.

Overexpression of Larp4B downregulates dMyc and reduces cell and organ sizes in Drosophila.

Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. Here we performed a gain-of-function screen and identified larp4B whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. We generated transgenic flies overexpressing wild-type Larp4B or a deletion variant lacking the LA and RRM domains, and demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. We found that the larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. Our results suggest that Larp4B is a negative regulator of dMyc.

Circular RNA_LARP4 inhibits cell proliferation and invasion of gastric cancer by sponging miR-424-5p and regulating LATS1 expression.

BACKGROUND: Non-coding RNAs (ncRNAs) have been shown to regulate gene expression involved in tumor progression of multiple malignancies. Our previous studies indicated that large tumor suppressor kinase 1 (LATS1), a core part of Hippo signaling pathway, functions as a tumor suppressor in gastric cancer (GC). But, the underlying molecular mechanisms by which ncRNAs modulate LATS1 expression in GC remain undetermined. METHODS: The correlation of LATS1 and has-miR-424-5p (miR-424) expression with clinicopathological characteristics and prognosis of GC patients was analyzed by TCGA RNA-sequencing data. A novel circular RNA_LARP4 (circLARP4) was identified to sponge miR-424 by circRNA expression profile and bioinformatic analysis. The binding site between miR-424 and LATS1 or circLARP4 was verified using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The expression and localization of circLARP4 in GC tissues were investigated by fluorescence in situ hybridization (FISH). MTT, colony formation, Transwell and EdU assays were performed to assess the effects of miR-424 or circLARP4 on cell proliferation and invasion. RESULTS: Increased miR-424 expression or decreased LATS1 expression was associated with pathological stage and unfavorable prognosis of GC patients. Ectopic expression of miR-424 promoted proliferation and invasion of GC cells by targeting LATS1 gene. Furthermore, circLARP4 was mainly localized in the cytoplasm and inhibited biological behaviors of GC cells by sponging miR-424. The expression of circLARP4 was downregulated in GC tissues and represented an independent prognostic factor for overall survival of GC patients. CONCLUSION: circLARP4 may act as a novel tumor suppressive factor and a potential biomarker in GC.

Cellular RNA-binding proteins LARP4 and PABPC1 synergistically facilitate viral translation of coronavirus PEDV.

Coronaviruses are causing epizootic diseases and thus are a substantial threat for both domestic and wild animals. These viruses depend on the host translation machinery to complete their life cycle. The current paper identified cellular RNA-binding proteins (RBPs), La-related protein 4 (LARP4) and polyadenylate-binding protein cytoplasmic 1 (PABPC1), as critical regulators of efficient translation of the coronavirus porcine epidemic diarrhea virus (PEDV) mRNA. In Vero cells, PEDV infection caused LARP4 to migrate from the nucleus to the cytoplasm in a chromosome region maintenance1 (CRM1)-independent pathway. In the absence of the nuclear export signal of LARP4, viral translation was not promoted by LARP4. A further study unveiled that the cytoplasmic LARP4 binds to the 3'-terminal untranslated region (3'UTR) of PEDV mRNA with the assistance of PABPC1 to facilitate viral translation. LARP4 knockdown reduced the promotion of the PABPC1-induced 3'UTR translation activity. Moreover, the rabbit reticulocyte lysate (RRL) system revealed that the prokaryotic expressed protein LARP4 and PABPC1 enhance PEDV mRNA translation. To our knowledge, this is the first study demonstrating that PEDV induces nucleo-cytoplasmic shuttling of LARP4 to enhance its own replication, which broadens our insights into how viruses use host's RBPs for the efficient translation of viral mRNA.

LARP4A and LARP4B in cancer: The new kids on the block.

Recent developments have mounted a stunning body of evidence underlying the importance of RNA binding proteins (RBPs) in cancer research. In this minireview we focus on LARP4A and LARP4B, two paralogs belonging to the superfamily of La-related proteins, and provide a critical overview of current research, including their roles in cancer pathogenesis and cell proliferation, migration, cell cycle and apoptosis. We highlight current controversies surrounding LARP4A and LARP4B and conclude that their complex roles in tumorigenesis are cell-, tissue- and context-dependent, warning that caution must be exercised before categorising either protein as an oncoprotein or tumour-suppressor. We also reveal that LARP4A and LARP4B have often been confused with one another, adding uncertainty in delineating their functions. We suggest that further functional and mechanistic studies of LARP4 proteins present significant challenges for future investigations to recognise the vital contributions of these RBPs in cancer research.

Interaction of LARP4 to filamin A mechanosensing domain regulates cell migrations.

Filamin A (FLNA) is an actin cross-linking protein that mediates mechanotransduction. Force-dependent conformational changes of FLNA molecule expose cryptic binding site of FLNA, allowing interaction with partners such as integrin, smoothelin, and fimbacin. Here, we identified La-related protein 4 (LARP4) as a new FLNA mechanobinding partner. LARP4 specifically interacts with the cleft formed by C and D strands of immunoglobulin-like repeat 21 (R21) which is blocked by A strand of R20 without force. We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro. We also determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 (F277A in human: F273A in mouse Larp4) that disrupts the interaction. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled LARP4 in living cells demonstrated that mutant LARP4 diffuses faster than WT LARP4. Proximity ligation assay (PLA) also confirmed their interaction and disruption of actin polymerization diminishes the interaction. Data mining of RNAseq analysis of LARP4 knockdown (KD) HEK293T cells suggested that LARP4 is involved in morphogenesis and cell motility. Consistent with this prediction, we found that KD of LARP4 increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells also leads to a higher cell migration speed compared to WT LARP4. These results demonstrated that the LARP4 interaction with FLNA regulates cell migration.

La-related protein 4 is enriched in vaccinia virus factories and is required for efficient viral replication in primary human fibroblasts.

In addition to the 3'-poly(A) tail, vaccinia virus mRNAs synthesized after viral DNA replication (post-replicative mRNAs) possess a 5'-poly(A) leader that confers a translational advantage in virally infected cells. These mRNAs are synthesized in viral factories, the cytoplasmic compartment where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, a previous study indicates that the poly(A)-binding protein (PABPC1)-which has a well-established role in RNA stability and translation-is absent in the viral factories. This prompts the question of whether other poly(A)-binding proteins engage vaccinia virus post-replicative mRNA in viral factories. Here, in this study, we found that La-related protein 4 (LARP4), a poly(A) binding protein, was enriched in viral factories in multiple types of cells during vaccinia virus infection. Further studies showed that LARP4 enrichment in the viral factories required viral post-replicative gene expression and functional decapping enzymes encoded by vaccinia virus. We further showed that knockdown of LARP4 expression in human foreskin fibroblasts (HFFs) reduced vaccinia virus DNA replication, post-replicative protein levels, and viral production. Interestingly, the knockdown of LARP4 expression also reduced protein levels from transfected mRNA containing a 5'-poly(A) leader in vaccinia virus-infected and uninfected HFFs. Taken together, our results identified a poly(A)-binding protein, LARP4, being enriched in the vaccinia virus viral factories and facilitating viral replication in HFFs. IMPORTANCE Vaccinia virus, the prototype poxvirus, encodes over 200 open reading frames (ORFs). Over 90 of vaccinia virus ORFs are transcribed post-viral DNA replication. All these mRNAs contain a 5'-poly(A) leader, as well as a 3'-poly(A) tail. They are synthesized in viral factories, where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, surprisingly, the poly(A) binding protein, PABPC1, that is important for mRNA metabolism and translation is not present in the viral factories, suggesting other poly(A) binding protein(s) may be present in viral factories. Here, we found another poly(A)-binding protein, La-related protein 4 (LARP4), enriched in viral factories during vaccinia virus infection. We also showed that LARP4 enrichment in the viral factories depends on viral post-replicative gene expression and functional viral decapping enzymes. The knockdown of LARP4 expression in human foreskin fibroblasts reduced vaccinia virus DNA replication, post-replicative gene expression, and viral production.

Circ_LARP4 regulates high glucose-induced cell proliferation, apoptosis, and fibrosis in mouse mesangial cells.

The current study aimed to investigate the role and underlying mechanisms of circ_LARP4 in diabetic nephropathy (DN). Here, mouse mesangial cells (SV40-MES13) were cultured with 30 mM glucose to establish a DN cellular model. The qRT-PCR results indicated that circ_LARP4 expression was downregulated in the DN cellular model compared to that in the control cells. As determined by an MTT assay, circ_LARP4 overexpression via the circ_LARP4 overexpression (OE) plasmids inhibited the cell proliferation rate. As determined by an Annexin V/PI kit and flow cytometry, circ_LARP4 overexpression increased the cell apoptosis rate. As measured by Western blot, circ_LARP4 overexpression enhanced BAX expression but reduced Bcl-2 expression, also suggesting an enhancement of cell apoptosis. Moreover, regarding cell fibrosis, circ_LARP4 overexpression reduced the mRNA levels of fibrosis markers, including fibronectin, collagen I and collagen IV. Interestingly, miR-424 was found to be reduced in the DN cellular model after transfection with the circ_LARP4 OE plasmids. In addition, restoration of miR-424 expression with the miR-424 mimics reversed the negative effects of circ_LARP4 overexpression on cell proliferation and fibrosis. In conclusion, circ_LARP4 was lower in the DN cellular model than in normal cells, and circ_LARP4 overexpression resulted in decreased cell proliferation and cell fibrosis but increased cell apoptosis in the DN cellular model by sponging miR-424.

Resonance assignment of human LARP4A La module.

Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a positive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La-module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La-module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisation of its La-module and here we report the assignment of 1H, 15N and 13C resonances resulting from our studies.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.