NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

lncRNA DLG1-AS1 promotes cervical cancer cell gemcitabine resistance by regulating miR-16-5p/HDGF.

AIM: To investigate the long non-coding RNA DLG1 Antisense RNA 1 (lncRNA DLG1-AS1) mechanism in cervical cancer cells with gemcitabine (GEM) resistance. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect DLG1-AS1, miR-16-5p, and hepatoma-derived growth factor (HDGF) expression in cervical cancer cells. The effects of DLG1-AS1 knockdown on cell viability, proliferation, and apoptosis were investigated in GEM-resistant cervical cancer cells. The binding of DLG1-AS1 with miR-16-5p and of miR-16-5p with HDGF was confirmed through dual-luciferase reporter assays. HDGF expression was detected through Western blotting. A xenograft model was established using stably transfected GEM-resistant cervical cancer cells to detect the role of DLG1-AS1 in tumorigenesis in vivo. RESULTS: DLG1-AS1 expression was significantly elevated in HeLa/GEM and SiHa/GEM cells. DLG1-AS1 silencing significantly reduced the viability and proliferation of GEM-resistant cervical cancer cells. DLG1-AS1 also promoted GEM sensitivity in cervical cancer cells by inhibiting miR-16-5p. Moreover, the tumor volume in nude mice in the DLG1-AS1 knockdown group decreased after GEM treatment. In addition, DLG1-AS1 targeted miR-16-5p, and miR-16-5p targeted HDGF. The miR-16-5p inhibitor reversed the DLG1-AS1 knockdown effect in GEM-resistant cervical cancer cells. CONCLUSION: Knockdown of DLG1-AS1 promoted GEM sensitivity in cervical cancer cells by regulating miR-16-5p/HDGF.

PDZ Proteins SCRIB and DLG1 Regulate Myeloma Cell Surface CD86 Expression, Growth, and Survival.

Despite advances in the treatment of multiple myeloma in the past decades, the disease remains incurable, and understanding signals and molecules that can control myeloma growth and survival are important for the development of novel therapeutic strategies. One such molecule, CD86, regulates multiple myeloma cell survival via its interaction with CD28 and signaling through its cytoplasmic tail. Although the CD86 cytoplasmic tail has been shown to be involved in drug resistance and can induce molecular changes in multiple myeloma cells, its function has been largely unexplored. Here, we show that CD86 cytoplasmic tail has a role in trafficking CD86 to the cell surface. This is due in part to a PDZ-binding motif at its C-terminus which is important for proper trafficking from the Golgi apparatus. BioID analysis revealed 10 PDZ domain-containing proteins proximal to CD86 cytoplasmic tail in myeloma cells. Among them, we found the planar cell polarity proteins, SCRIB and DLG1, are important for proper CD86 surface expression and the growth and survival of myeloma cells. These findings indicate a mechanism by which myeloma cells confer cellular survival and drug resistance and indicate a possible motif to target for therapeutic gain. IMPLICATIONS: These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease.

The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p.

BACKGROUND: Dysregulation of circular RNAs (circRNAs) plays an important role in the development of gastric cancer; thus, revealing the biological and molecular mechanisms of abnormally expressed circRNAs is critical for identifying novel therapeutic targets in gastric cancer. METHODS: A circRNA microarray was performed to identify differentially expressed circRNAs between primary and distant metastatic tissues and between gastric cancer tissues sensitive or resistant to anti-programmed cell death 1 (PD-1) therapy. The expression of circRNA discs large homolog 1 (DLG1) was determined in a larger cohort of primary and distant metastatic gastric cancer tissues. The role of circDLG1 in gastric cancer progression was evaluated both in vivo and in vitro, and the effect of circDLG1 on the antitumor activity of anti-PD-1 was evaluated in vivo. The interaction between circDLG1 and miR-141-3p was assessed by RNA immunoprecipitation and luciferase assays. RESULTS: circDLG1 was significantly upregulated in distant metastatic lesions and gastric cancer tissues resistant to anti-PD-1 therapy and was associated with an aggressive tumor phenotype and adverse prognosis in gastric cancer patients treated with anti-PD-1 therapy. Ectopic circDLG1 expression promoted the proliferation, migration, invasion, and immune evasion of gastric cancer cells. Mechanistically, circDLG1 interacted with miR-141-3p and acted as a miRNA sponge to increase the expression of CXCL12, which promoted gastric cancer progression and resistance to anti-PD-1-based therapy. CONCLUSIONS: Overall, our findings demonstrate how circDLG1 promotes gastric cancer cell proliferation, migration, invasion and immune evasion and provide a new perspective on the role of circRNAs during gastric cancer progression.

Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Roles of miR-204 in retinal development and maintenance.

The retina is the innermost part of the eye of most vertebrates and it is essential for vision. The development, maintenance, and function of this laminated structure is tightly regulated by numerous genes. Deficiencies in the expression of these genes as well as deregulation of various molecular mechanisms can cause retinopathies and blindness. MicroRNAs (miRNAs) are one of the most important and effective molecular regulatory mechanisms that underlie the biology of the retina. miRNAs have specific functional roles in the development and maintenance of different retinal layers and retinal cell types. While previous studies have reported a large number of miRNAs linked to development, maintenance and diseases of the retina, no comprehensive study has properly discussed and integrated data from these studies. Given the particular importance of miR-204 in retinal biology, we intend to critically discuss the expression and functional significance of this miRNA in the development, maintenance, and pathologies of the retina. Moreover, we explore biological processes through which miR-204 influences retinal pathophysiology. This review highlights the crucial functions of miR-204 in the retina and suggests the putative mechanism of miR-204 action in retinal biology.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Scrib and Dlg1 polarity proteins regulate Ag presentation in human dendritic cells.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

HPV E6 and E7 oncoproteins cooperatively alter the expression of Disc Large 1 polarity protein in epithelial cells.

BACKGROUND: Persistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions. METHODS: Due to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively. RESULTS: We demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation. CONCLUSIONS: In general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.

Altered expression of DLG1-AS1 distinguished papillary thyroid carcinoma from benign thyroid nodules.

BACKGROUND: Benign thyroid nodules (BTN) are frequently diagnosed as papillary thyroid carcinoma (PTC), leading to unnecessary treatment. We found that plasma lncRNA DLG1-AS1 was upregulated in PTC patients but not in BTN patients and healthy controls. METHODS: In this study DLG1-AS1 and miR-199a-3p in plasma of both PTC patients and BTN patients were detected by qPCR. ROC curve analysis was performed for diagnostic analysis. Overexpression experiments were performed to analyze the interaction between DLG1-AS1 and miR-199a-3p. CCK-8 assay was performed to analyze cell proliferation. RESULTS: In this study, upregulation of DLG1-AS1 distinguished PTC patients from BTN patients and healthy controls. Plasma miR-199a-3p was downregulated in PTC patients compared with healthy controls and BTN patients. Plasma levels of miR-199a-3p were inversely correlated in PTC patients, but not in BTN patients and healthy controls. miR-199a-3p overexpression failed to significantly affect DLG1-AS1, while DLG1-AS1 overexpression resulted in downregulated miR-199a-3p, In addition, DLG1-AS1 overexpression promoted the proliferation of PTC cells. miR-199a-3p overexpression played an opposite role and attenuated the effects of DLG1-AS1 overexpression. CONCLUSIONS: Therefore, DLG1-AS1 may promote PTC by downregulating miR-199a-3p.

Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK1 cells.

Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, "GTKA," is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2. The role of PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC-PK1 renal collecting cells by confocal microscopy and cell surface biotinylation techniques. These experiments indicated that distribution of AQP2 and SAP97 overlapped in the kidneys and LLC-PK1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser256, plays an important role in the translocation of AQP2 in the kidney.

Epigenetic Regulation of Dlg1, via Kaiso, Alters Mitotic Spindle Polarity and Promotes Intestinal Tumorigenesis.

Both alterations to the epigenome and loss of polarity have been linked to cancer initiation, progression, and metastasis. It has previously been demonstrated that loss of the epigenetic reader protein Kaiso suppresses intestinal tumorigenesis in the Apc+/min mouse model, in which altered polarity plays a key role. Thus, we investigated the link between Kaiso deficiency, polarity, and suppression of intestinal tumorigenesis. We used Kaiso-deficient mice to conditionally delete Apc within the intestinal epithelia and demonstrated upregulation of the spindle polarity genes Dlg1 and Dlgap1. To understand the role of Dlg1, we generated Villin-creApc+/minDlg1flx/flx Kaiso-/y mice to analyze gene expression, survival, tumor burden, and spindle orientation. In vivo analysis of the Dlg1-deficient intestine revealed improper orientation of mitotic spindles and a decreased rate of cellular migration. Loss of Dlg1 decreased survival in Apc+/min mice, validating its role as a tumor suppressor in the intestine. Significantly, the increased survival of Apc+/minKaisoy/- mice was shown to be dependent on Dlg1 expression. Taken together, these data indicate that maintenance of spindle polarity in the intestinal crypt requires appropriate regulation of Dlg1 expression. As Dlg1 loss leads to incorrect spindle orientation and a delay in cells transiting the intestinal crypt. We propose that the delayed exit from the crypt increase the window in which spontaneous mutations can become fixed, producing a "tumor-permissive" environment, without an increase in mutation rate. IMPLICATIONS: Loss of mitotic spindle polarity delays the exit of cells from the intestinal crypt and promotes a tumorigenic environment.

Differential expression of DLG1 as a common trait in different human diseases: an encouraging issue in molecular pathology.

Human disc large (DLG1) is a scaffolding protein that through the interaction with diverse cell partners participates in the control of key cellular processes such as polarity, proliferation and migration. Experimental data have mainly identified DLG1 as a tumor suppressor. An outstanding point for DLG1 protein is that altered DLG1 expression and DLG1 gene mutations were observed in different pathologies, including cancer and neurological and immunological disorders. Evident changes in DLG1 abundance and/or cell localization were identified in a number of studies suggesting its participation in molecular mechanisms responsible for the development of such illnesses. In this review, we focus on some of the latest findings regarding DLG1 alterations in different diseases as well as its potential use as a biomarker for pathological progression. We further address the current knowledge on the molecular mechanisms regulating DLG1 expression and the posttranslational modifications that may affect DLG1 cell localization and functions. Despite the advances in this field, there are still open questions about the precise molecular link between alterations in DLG1 expression and the development of each specific pathology. The complete understanding of this concern will give us new scenarios for the design of promising diagnosis and therapeutic tools.