Graph Convolutional Network for predicting secondary structure of RNA.

The prediction of RNA secondary structures is essential for understanding its underlying principles and applications in diverse fields, including molecular diagnostics and RNA-based therapeutic strategies. However, the complexity of the search space presents a challenge. This work proposes a Graph Convolutional Network (GCNfold) for predicting the RNA secondary structure. GCNfold considers an RNA sequence as graph-structured data and predicts posterior base-pairing probabilities given the prior base-pairing probabilities, calculated using McCaskill's partition function. The performance of GCNfold surpasses that of the state-of-the-art folding algorithms, as we have incorporated minimum free energy information into the richly parameterized network, enhancing its robustness in predicting non-homologous RNA secondary structures. A Symmetric Argmax Post-processing algorithm ensures that GCNfold formulates valid structures. To validate our algorithm, we applied it to the SARS-CoV-2 E gene and determined the secondary structure of the E-gene across the Betacoronavirus subgenera.

Aggregation determines the selectivity of membrane-active anticancer and antimicrobial peptides: The case of killerFLIP.

Host defense peptides selectively kill bacterial and cancer cells (including those that are drug-resistant) by perturbing the permeability of their membranes, without being significantly toxic to the host. Coulombic interactions between these cationic and amphipathic peptides and the negatively charged membranes of pathogenic cells contribute to the selective toxicity. However, a positive charge is not sufficient for selectivity, which can be achieved only by a finely tuned balance of electrostatic and hydrophobic driving forces. A common property of amphipathic peptides is the formation of aggregated structures in solution, but the role of this phenomenon in peptide activity and selectivity has received limited attention. Our data on the anticancer peptide killerFLIP demonstrate that aggregation strongly increases peptide selectivity, by reducing the effective peptide hydrophobicity and thus the affinity towards membranes composed of neutral lipids (like the outer layer of healthy eukaryotic cell membranes). Aggregation is therefore a useful tool to modulate the selectivity of membrane active peptides and peptidomimetics.

A Peroxidase Peroxiredoxin 1-Specific Redox Regulation of the Novel FOXO3 microRNA Target let-7.

Precision in redox signaling is attained through posttranslational protein modifications such as oxidation of protein thiols. The peroxidase peroxiredoxin 1 (PRDX1) regulates signal transduction through changes in thiol oxidation of its cysteines. We demonstrate here that PRDX1 is a binding partner for the tumor suppressive transcription factor FOXO3 that directly regulates the FOXO3 stress response. Heightened oxidative stress evokes formation of disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Absence of PRDX1 enhances FOXO3 nuclear localization and transcription that are dependent on the presence of Cys31 or Cys150 within FOXO3. Notably, FOXO3-T32 phosphorylation is constitutively enhanced in these mutants, but nuclear translocation of mutant FOXO3 is restored with PI3K inhibition. Here we show that on H2O2 exposure, transcription of tumor suppressive miRNAs let-7b and let-7c is regulated by FOXO3 or PRDX1 expression levels and that let-7c is a novel target for FOXO3. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. Altogether, these data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling axis that fine tunes FOXO3 activity toward the transcription of gene targets in response to oxidative stress. Antioxid. Redox Signal. 28, 62-77.

Changes in membrane lipids drive increased endocytosis following Fas ligation.

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles.

Meta-analysis of transcriptome data identifies a novel 5-gene pancreatic adenocarcinoma classifier.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is largely incurable due to late diagnosis. Superior early detection biomarkers are critical to improving PDAC survival and risk stratification. EXPERIMENTAL DESIGN: Optimized meta-analysis of PDAC transcriptome datasets identified and validated key PDAC biomarkers. PDAC-specific expression of a 5-gene biomarker panel was measured by qRT-PCR in microdissected patient-derived FFPE tissues. Cell-based assays assessed impact of two of these biomarkers, TMPRSS4 and ECT2, on PDAC cells. RESULTS: A 5-gene PDAC classifier (TMPRSS4, AHNAK2, POSTN, ECT2, SERPINB5) achieved on average 95% sensitivity and 89% specificity in discriminating PDAC from non-tumor samples in four training sets and similar performance (sensitivity = 94%, specificity = 89.6%) in five independent validation datasets. This classifier accurately discriminated PDAC from chronic pancreatitis (AUC = 0.83), other cancers (AUC = 0.89), and non-tumor from PDAC precursors (AUC = 0.92) in three independent datasets. Importantly, the classifier distinguished PanIN from healthy pancreas in the PDX1-Cre;LSL-KrasG12D PDAC mouse model. Discriminatory expression of the PDAC classifier genes was confirmed in microdissected FFPE samples of PDAC and matched surrounding non-tumor pancreas or pancreatitis. Notably, knock-down of TMPRSS4 and ECT2 reduced PDAC soft agar growth and cell viability and TMPRSS4 knockdown also blocked PDAC migration and invasion. CONCLUSIONS: This study identified and validated a highly accurate 5-gene PDAC classifier for discriminating PDAC and early precursor lesions from non-malignant tissue that may facilitate early diagnosis and risk stratification upon validation in prospective clinical trials. Cell-based experiments of two overexpressed proteins encoded by the panel, TMPRSS4 and ECT2, suggest a causal link to PDAC development and progression, confirming them as potential therapeutic targets.

A novel caspase 8 selective small molecule potentiates TRAIL-induced cell death.

Recombinant soluble TRAIL and agonistic antibodies against TRAIL receptors (DR4 and DR5) are currently being created for clinical cancer therapy, due to their selective killing of cancer cells and high safety characteristics. However, resistance to TRAIL and other targeted therapies is an important issue facing current cancer research field. An attractive strategy to sensitize resistant malignancies to TRAIL-induced cell death is the design of small molecules that target and promote caspase 8 activation. For the first time, we describe the discovery and characterization of a small molecule that directly binds caspase 8 and enhances its activation when combined with TRAIL, but not alone. The molecule was identified through an in silico chemical screen for compounds with affinity for the caspase 8 homodimer's interface. The compound was experimentally validated to directly bind caspase 8, and to promote caspase 8 activation and cell death in single living cells or population of cells, upon TRAIL stimulation. Our approach is a proof-of-concept strategy leading to the discovery of a novel small molecule that not only stimulates TRAIL-induced apoptosis in cancer cells, but may also provide insights into the structure-function relationship of caspase 8 homodimers as putative targets in cancer.

PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor.

FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ's development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor.

Combination of bortezomib and mitotic inhibitors down-modulate Bcr-Abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant Bcr-Abl-positive leukemic cells.

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.

Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP.

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Harnessing the tumor suppressor function of FOXO as an alternative therapeutic approach in cancer.

The promotion of cellular survival, dedifferentiation, and uncontrolled proliferation via the suppression of apoptotic effectors is a fundamental characteristic of tumor cells. As substrates that are negatively regulated by oncogenic signaling cascades driven by AKT, SGK (serum- and glucocorticoid-inducible kinase), IkB kinase (IKK), ERK, and cyclin-dependent kinases (CDK), forkhead box-class O (FOXO) transcription factors have emerged as bona fide tumor suppressors. These transcription factors indeed regulate a variety of cellular responses and themselves are regulated by reversible phosphorylation, acetylation, ubiquitination and miRNAs. This review will discuss our current understanding of mechanisms for FOXO regulation and the potential implications for therapeutically restoring FOXO transcriptional activity.

Bortezomib treatment causes remission in a Ph+ALL patient and reveals FoxO as a theranostic marker.

BCR-ABL is a key mediator in the pathogenesis of all cases of chronic myelogenous leukemia (CML) and a subset of precursor B-acute lymphoblastic leukemia (Ph+ALL). Previous animal and cell-based studies have shown that the expression of members of the Forkhead family of tumor suppressors, including FoxO3, is suppressed in BCR-ABL-expressing cells. Furthermore, it has been reported that the proteasomal degradation pathway plays an important role in suppression of FoxO expression in BCR-ABL-transformed cells. In this study, a patient diagnosed with Ph+ALL and refractory to standard therapies was treated with a proteasome inhibitor (bortezomib)-based chemotherapy regimen. This treatment resulted in complete hematologic, cytogenetic and molecular remission with excellent performance status for > 4 years since her initial diagnosis. FoxO3 was not detectable within the blasts of this patient at diagnosis and was 'rescued' following treatment with bortezomib therapy, leading to her recovery. As a next step, in the attempt to propose FoXO3 as a therapeutic target and a theranostic marker, we further validated FoxO3 expression in human bone marrow biopsy samples. Human core biopsy samples of Ph+ALL and Ph-negative-negative ALL, along with uninvolved controls, revealed that FoxO3 down-regulation was specific to Ph+ALL. This study provides support that FoxO3 is a good biomarker for BCR-ABL-mediated leukemogenesis. Additionally, proteasomal inhibition by bortezomib may be a promising therapeutic option in Philadelphia-positive ALL, where FoxO3 is downregulated.

Apoptotic cell signaling in cancer progression and therapy.

Apoptosis is a tightly regulated cell suicide program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Impairment of this native defense mechanism promotes aberrant cellular proliferation and the accumulation of genetic defects, ultimately resulting in tumorigenesis, and frequently confers drug resistance to cancer cells. The regulation of apoptosis at several levels is essential to maintain the delicate balance between cellular survival and death signaling that is required to prevent disease. Complex networks of signaling pathways act to promote or inhibit apoptosis in response to various cues. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Various upstream signaling pathways can modulate apoptosis by converging on, and thereby altering the activity of, common central control points within the apoptotic signaling pathways, which involve the BCL-2 family proteins, inhibitor of apoptosis (IAP) proteins, and FLICE-inhibitory protein (c-FLIP). This review highlights the role of these fundamental regulators of apoptosis in the context of both normal apoptotic signaling mechanisms and dysregulated apoptotic pathways that can render cancer cells resistant to cell death. In addition, therapeutic strategies aimed at modulating the activity of BCL-2 family proteins, IAPs, and c-FLIP for the targeted induction of apoptosis are briefly discussed.

Discovery of a small-molecule type II inhibitor of wild-type and gatekeeper mutants of BCR-ABL, PDGFRalpha, Kit, and Src kinases: novel type II inhibitor of gatekeeper mutants.

Many clinically validated kinases, such as BCR-ABL, c-Kit, PDGFR, and EGFR, become resistant to adenosine triphosphate-competitive inhibitors through mutation of the so-called gatekeeper amino acid from a threonine to a large hydrophobic amino acid, such as an isoleucine or methionine. We have developed a new class of adenosine triphosphate competitive inhibitors, exemplified by HG-7-85-01, which is capable of inhibiting T315I- BCR-ABL (clinically observed in chronic myeloid leukemia), T670I-c-Kit (clinically observed in gastrointestinal stromal tumors), and T674I/M-PDGFRalpha (clinically observed in hypereosinophilic syndrome). HG-7-85-01 is unique among all currently reported kinase inhibitors in having the ability to accommodate either a gatekeeper threonine, present in the wild-type forms of these kinases, or a large hydrophobic amino acid without becoming a promiscuous kinase inhibitor. The distinctive ability of HG-7-85-01 to simultaneously inhibit both wild-type and mutant forms of several kinases of clinical relevance is an important step in the development of the next generation of tyrosine kinase inhibitors.

Protein phosphatase 2A reactivates FOXO3a through a dynamic interplay with 14-3-3 and AKT.

Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation.

Proteasome inhibition causes regression of leukemia and abrogates BCR-ABL-induced evasion of apoptosis in part through regulation of forkhead tumor suppressors.

BCR-ABL plays an essential role in the pathogenesis of chronic myeloid leukemia (CML) and some cases of acute lymphocytic leukemia (ALL). Although ABL kinase inhibitors have shown great promise in the treatment of CML, the persistence of residual disease and the occurrence of resistance have prompted investigations into the molecular effectors of BCR-ABL. Here, we show that BCR-ABL stimulates the proteasome-dependent degradation of members of the forkhead family of tumor suppressors in vitro, in an in vivo animal model, and in samples from patients with BCR-ABL-positive CML or ALL. As several downstream mediators of BCR-ABL are regulated by the proteasome degradation pathway, we also show that inhibition of this pathway, using bortezomib, causes regression of CML-like disease. Bortezomib treatment led to inhibition of BCR-ABL-induced suppression of FoxO proteins and their proapoptotic targets, tumor necrosis factor-related apoptosis-inducing ligand and BIM, thereby providing novel insights into the molecular effects of proteasome inhibitor therapy. We additionally show sensitivity of imatinib-resistant BCR-ABL T315I cells to bortezomib. Our data delineate the involvement of FoxO proteins in BCR-ABL-induced evasion of apoptosis and provide evidence that bortezomib is a candidate therapeutic in the treatment of BCR-ABL-induced leukemia.

A double hit to kill tumor and endothelial cells by TRAIL and antiangiogenic 3TSR.

As tumor development relies on a coordination of angiogenesis and tumor growth, an efficient antitumor strategy should target both the tumor and its associated vessels. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner. Additionally, thrombospondin-1, a naturally occurring inhibitor of angiogenesis, and a recombinant protein containing functional domains of thrombospondin-1, 3TSR, have been shown to be necessary and sufficient to inhibit tumor angiogenesis. Here, we show that a combination of a TRAIL receptor 2 agonist antibody, Lexatumumab, and 3TSR results in a significantly enhanced and durable tumor inhibition. We further observed that 3TSR induces apoptosis in primary endothelial cells by up-regulating the expression of TRAIL receptors 1 and 2 in a CD36 and Jun NH(2)-terminal kinase-dependent manner leading to the activation of both intrinsic and extrinsic apoptotic machineries. The modulation of these pathways is critical for 3TSR-induced apoptosis as disrupting either via specific inhibitors reduced apoptosis. Moreover, 3TSR attenuates the Akt survival pathway. These studies indicate that 3TSR plays a critical role in regulating the proapoptotic signaling pathways that control growth and death in endothelial cells and that a combination of TRAIL and 3TSR acts as a double hit against tumor and tumor-associated vessels.

Fas death receptor enhances endocytic membrane traffic converging into the Golgi region.

The death receptor Fas/CD95 initiates apoptosis by engaging diverse cellular organelles including endosomes. The link between Fas signaling and membrane traffic has remained unclear, in part because it may differ in diverse cell types. After a systematic investigation of all known pathways of endocytosis, we have clarified that Fas activation opens clathrin-independent portals in mature T cells. These portals drive rapid internalization of surface proteins such as CD59 and depend upon actin-regulating Rho GTPases, especially CDC42. Fas-enhanced membrane traffic invariably produces an accumulation of endocytic membranes around the Golgi apparatus, in which recycling endosomes concentrate. This peri-Golgi polarization has been documented by colocalization analysis of various membrane markers and applies also to active caspases associated with internalized receptor complexes. Hence, T lymphocytes show a diversion in the traffic of endocytic membranes after Fas stimulation that seems to resemble the polarization of membrane traffic after their activation.

Analysis of TNF-related apoptosis-inducing ligand in vivo through bone marrow transduction and transplantation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines. TRAIL has gained much attention because of its ability to preferentially kill tumor cells with no apparent toxic side effects. Recently, different TRAIL receptor agonists, including TRAIL itself and various agonistic monoclonal antibodies against the two apoptosis-inducing human TRAIL receptors, have been developed as novel cancer therapeutics and are currently under investigation in clinical trials. However, the mechanisms by which TRAIL mediates its selective antineoplastic activity are still not well understood. In addition to playing a role in cancer immune surveillance and tumor suppression, TRAIL has been associated with immune homeostasis, inflammatory diseases, and autoimmunity. In light of the multifunctional role of TRAIL in mediating various pathologic conditions and the potential benefits of TRAIL-based therapies, the study of the physiologic significance of TRAIL is of great importance. Here, we describe a syngeneic system for the characterization of the in vivo function of TRAIL. By use of this model, in which the full-length murine TRAIL protein is overexpressed in the hematopoietic cells of wild-type mice, the in vivo tumoricidal activity of TRAIL overexpression can be studied on syngeneic murine tumor cell challenge, and the potential toxicity of TRAIL protein to normal tissues can also be analyzed.