The Role of Toll-like Receptors (TLRs) Mediated Inflammation in Pancreatic Cancer Pathophysiology.

Pancreatic cancer (PC) is one of the most lethal forms of cancer, characterized by its aggressiveness and metastatic potential. Despite significant improvements in PC treatment and management, the complexity of the molecular pathways underlying its development has severely limited the available therapeutic opportunities. Toll-like receptors (TLRs) play a pivotal role in inflammation and immune response, as they are involved in pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Activation of TLRs initiates a signaling cascade, which in turn, leads to the transcription of several genes involved in inflammation and anti-microbial defense. TLRs are also deregulated in several cancers and can be used as prognostic markers and potential targets for cancer-targeted therapy. In this review we discuss the current knowledge about the role of TLRs in PC progression, focusing on the available TLRs-targeting compounds and their possible use in PC therapy.

UBAC1/KPC2 Regulates TLR3 Signaling in Human Keratinocytes through Functional Interaction with the CARD14/CARMA2sh-TANK Complex.

CARD14/CARMA2 is a scaffold molecule whose genetic alterations are linked to human inherited inflammatory skin disorders. However, the mechanisms through which CARD14/CARMA2 controls innate immune response and chronic inflammation are not well understood. By means of a yeast two-hybrid screening, we identified the UBA Domain Containing 1 (UBAC1), the non-catalytic subunit of the E3 ubiquitin-protein ligase KPC complex, as an interactor of CARMA2sh, the CARD14/CARMA2 isoform mainly expressed in human keratinocytes. UBAC1 participates in the CARMA2sh/TANK complex and promotes K63-linked ubiquitination of TANK. In human keratinocytes, UBAC1 negatively regulates the NF-κF-activating capacity of CARMA2sh following exposure to poly (I:C), an agonist of Toll-like Receptor 3. Overall, our data indicate that UBAC1 participates in the inflammatory signal transduction pathways involving CARMA2sh.

Adapting and Surviving: Intra and Extra-Cellular Remodeling in Drug-Resistant Gastric Cancer Cells.

Despite the significant recent advances in clinical practice, gastric cancer (GC) represents a leading cause of cancer-related deaths in the world. In fact, occurrence of chemo-resistance still remains a daunting hindrance to effectiveness of the current approach to GC therapy. There is accumulating evidence that a plethora of cellular and molecular factors is implicated in drug-induced phenotypical switching of GC cells. Among them, epithelial-mesenchymal transition (EMT), autophagy, drug detoxification, DNA damage response and drug target alterations, have been reported as major determinants. Intriguingly, resistant GC phenotype may be the result of GC cell-induced tumor microenvironment (TME) remodeling, which is currently emerging as a key player in promoting drug resistance and overcoming cytotoxic effects of drugs. In this review, we discuss the possible mechanisms of drug resistance and their involvement in determining current GC therapies failure.

Exploring the Molecular Crosstalk between Pancreatic Bud and Mesenchyme in Embryogenesis: Novel Signals Involved.

Pancreatic organogenesis is a multistep process that requires the cooperation of several signaling pathways. In this context, the role of pancreatic mesenchyme is important to define the epithelium development; nevertheless, the precise space-temporal signaling activation still needs to be clarified. This study reports a dissection of the pancreatic embryogenesis, highlighting the molecular network surrounding the epithelium-mesenchyme interaction. To investigate this crosstalk, pancreatic epithelium and surrounding mesenchyme, at embryonic day 10.5, were collected through laser capture microdissection (LCM) and characterized based on their global gene expression. We performed a bioinformatic analysis to hypothesize crosstalk interactions, validating the most promising genes and verifying the precise localization of their expression in the compartments, by RNA in situ hybridization (ISH). Our analyses pointed out also the c-Met gene, a very well-known factor involved in stimulating motility, morphogenesis, and organ regeneration. We also highlighted the potential crosstalk between Versican (Vcan) and Syndecan4 (Sdc4) since these genes are involved in pancreatic tissue repair, strengthening the concept that the same signaling pathways required during pancreatic embryogenesis are also involved in tissue repair. This finding leads to novel strategies for obtaining functional pancreatic stem cells for cell replacement therapies.

NF-κB Essential Modulator (NEMO) Is Critical for Thyroid Function.

The I-κB kinase (IKK) subunit NEMO/IKKγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology.

The E3 Ubiquitin Ligase RNF7 Negatively Regulates CARD14/CARMA2sh Signaling.

The three CARD-containing MAGUK (CARMA) proteins function as scaffolding molecules that regulate activation of the pro-inflammatory transcription factor NF-κB. Recently, mutations in CARMA2 have been linked to psoriasis susceptibility due to their acquired altered capacity to activate NF-κB. By means of two-hybrid screening with yeast, we identified RING finger protein 7 (RNF7) as an interactor of CARMA2. We present evidence that RNF7 functions as a negative regulator of the NF-κB-activating capacity of CARMA2. Mechanistically, RNF7 influences CARMA2 signaling by regulating the ubiquitination state of MALT1 and the NF-κB-regulatory molecule NEMO. Interestingly, CARMA2short (CARMA2sh) mutants associated with psoriasis susceptibility escape the negative control exerted by RNF7. In conclusion, our findings identify a new mechanism through which the ability of CARMA2 to activate NF-κB is regulated, which could have significant implications for our understanding of why mutations of this protein trigger human psoriasis.

Role of IL-6/STAT3 Axis in Resistance to Cisplatin in Gastric Cancers.

Gastric cancer, the second most common cause of death worldwide, is characterized by poor prognosis and low responsiveness to chemotherapy. Indeed, multidrug resistance, based mainly on cellular and molecular factors, remains one of the most limiting factors of the current approach to gastric cancer (GC) therapy. We employed a comprehensive gene expression analysis through data mining of publicly available databases to assess the role of the signal transducer and activator of transcription 3 (STAT3) in gastric cancer drug efficiency. It has been proposed that gastric cancer cells are less sensitive to these drugs because they develop resistance to these agents through activating alternative signalling pathways responsible for overcoming pharmacological inhibition. Our study evaluated the hypothesis that activating STAT3 signalling in response to cisplatin reduces the reaction to the drug. Consistent with this hypothesis, inhibition of interleukin 6 (IL-6)/STAT3 in combination therapy with cisplatin prevented both STAT3 activation and more lethality than induction by a single agent. The data suggest that the IL-6/STAT3 axis block associated with cisplatin treatment may represent a strategy to overcome resistance.

Gene Regulatory Network Characterization of Gastric Cancer's Histological Subtypes: Distinctive Biological and Clinically Relevant Master Regulators.

Gastric cancer (GC) molecular heterogeneity represents a major determinant for clinical outcomes, and although new molecular classifications have been introduced, they are not easy to translate from bench to bedside. We explored the data from GC public databases by performing differential gene expression analysis (DEGs) and gene network reconstruction to identify master regulators (MRs), as well as a gene set analysis (GSA) to reveal their biological features. Moreover, we evaluated the association of MRs with clinicopathological parameters. According to the GSA, the Diffuse group was characterized by an epithelial-mesenchymal transition (EMT) and inflammatory response, while the Intestinal group was associated with a cell cycle and drug resistance pathways. In particular, the regulons of Diffuse MRs, such as Vgll3 and Ciita, overlapped with the EMT and interferon-gamma response, while the regulons Top2a and Foxm1 were shared with the cell cycle pathways in the Intestinal group. We also found a strict association between MR activity and several clinicopathological features, such as survival. Our approach led to the identification of genes and pathways differentially regulated in the Intestinal and Diffuse GC histotypes, highlighting biologically interesting MRs and subnetworks associated with clinical features and prognosis, suggesting putative actionable candidates.

Gastric Cancer Stem Cells: A Glimpse on Metabolic Reprogramming.

Gastric cancer (GC) is one of the most widespread causes of cancer-related death worldwide. Recently, emerging implied that gastric cancer stem cells (GCSCs) play an important role in the initiation and progression of GC. This subpopulation comprises cells with several features, such as self-renewal capability, high proliferating rate, and ability to modify their metabolic program, which allow them to resist current anticancer therapies. Metabolic pathway intermediates play a pivotal role in regulating cell differentiation both in tumorigenesis and during normal development. Thus, the dysregulation of both anabolic and catabolic pathways constitutes a significant opportunity to target GCSCs in order to eradicate the tumor progression. In this review, we discuss the current knowledge about metabolic phenotype that supports GCSC proliferation and we overview the compounds that selectively target metabolic intermediates of CSCs that can be used as a strategy in cancer therapy.

Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells.

Gastric cancer (GC) is characterized by poor efficacy and modest clinical impact of current therapies, in which apoptosis evasion is relevant. Intracellular calcium homeostasis dysregulation is associated with apoptosis escaping, and aberrant expression of calcium regulator genes could promote GC drug resistance. Since we previously found a prognostic value for TRPV2 calcium channel expression in GC, we aimed to characterize the role of TRPV2 in cisplatin resistance. Using the TCGA-STAD dataset, we performed a differential gene expression analysis between GC samples in upper and lower tertiles of TRPV2 expression, and then through a gene set analysis, we highlighted the enriched ontology and canonical pathways. We used qRT-PCR to assess TRPV2 expression in three GC cell lines and flow cytometry to evaluate cisplatin-induced cell death rates. Calcium green-1-AM assay was used to estimate differences in intracellular Ca2+ concentrations after inhibition of TRPV2. We engineered AGS cell line to overexpress TRPV2 and used confocal microscopy to quantify its overexpression and localization and flow cytometry to evaluate their sensitivity to cisplatin. Consistent with our hypothesis, among enriched gene sets, we found a significant number of those involved in the regulation of apoptosis. Subsequently, we found an inverse correlation between TRPV2 expression and sensitivity to cisplatin in GC cell lines. Moreover, we demonstrated that inhibition of TRPV2 activity by tranilast blocks the efflux of Ca2+ ions and, in combination with cisplatin, induced a significant increase of apoptotic cells (p = 0.004). We also demonstrated that TRPV2 exogenous expression confers a drug-resistant phenotype, and that tranilast is able to revert this phenotype, restoring cisplatin sensitivity. Our findings consistently suggested that TRPV2 could be a potential target for overcoming cisplatin resistance by promoting apoptosis. Notably, our data are a prerequisite for the potential reposition of tranilast to the treatment of GC patients and anticipate the in vivo evaluation.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

Retinoic Acid Induces Embryonic Stem Cells (ESCs) Transition to 2 Cell-Like State Through a Coordinated Expression of Dux and Duxbl1.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) of the blastocyst. In serum/LIF culture condition, they show variable expression of pluripotency genes that mark cell fluctuation between pluripotency and differentiation metastate. The ESCs subpopulation marked by zygotic genome activation gene (ZGA) signature, including Zscan4, retains a wider differentiation potency than epiblast-derived ESCs. We have recently shown that retinoic acid (RA) significantly enhances Zscan4 cell population. However, it remains unexplored how RA initiates the ESCs to 2-cell like reprogramming. Here we found that RA is decisive for ESCs to 2C-like cell transition, and reconstructed the gene network surrounding Zscan4. We revealed that RA regulates 2C-like population co-activating Dux and Duxbl1. We provided novel evidence that RA dependent ESCs to 2C-like cell transition is regulated by Dux, and antagonized by Duxbl1. Our suggested mechanism could shed light on the role of RA on ESC reprogramming.

CARMA2sh and ULK2 control pathogen-associated molecular patterns recognition in human keratinocytes: psoriasis-linked CARMA2sh mutants escape ULK2 censorship.

The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.

Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

Functional characterization of zebrafish (Danio rerio) Bcl10.

The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation.

TRAF6-mediated ubiquitination of NEMO requires p62/sequestosome-1.

The atypical protein kinase C-interacting protein p62/sequestosome-1 (p62) has emerged as a crucial molecule in a variety of cellular functions due to its involvement in various signaling mechanisms. p62 has been implicated in the activation of NF-κB in TNFα-stimulated cells and has been shown to be activated in response to interleukin-1ß (IL-1ß). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-κB transcription factor. Depletion of p62 obtained through a short interfering RNA targeting p62 mRNA abrogated TRAF6 capacity to promote NEMO ubiquitination and severely impairs NF-κB activation following IL-1ß stimulation. Together, these results indicate that p62 is an important intermediary in the NF-κB activation pathways implemented through non-degradative ubiquitination events.

The Dishevelled, EGL-10 and pleckstrin (DEP) domain-containing protein DEPDC7 binds to CARMA2 and CARMA3 proteins, and regulates NF-κB activation.

The molecular complexes containing BCL10, MALT1 and CARMA proteins (CBM complex) have been recently identified as a key component in the signal transduction pathways that regulate activation of Nuclear Factor kappaB (NF-κB) transcription factor. Herein we identified the DEP domain-containing protein DEPDC7 as cellular binding partners of CARMA2 and CARMA3 proteins. DEPDC7 displays a cytosolic distribution and its expression induces NF-κB activation. Conversely, shRNA-mediated abrogation of DEPDC7 results in impaired NF-κB activation following G protein-coupled receptors stimulation, or stimuli that require CARMA2 and CARMA3, but not CARMA1. Thus, this study identifies DEPDC7 as a CARMA interacting molecule, and provides evidence that DEPDC7 may be required to specifically convey on the CBM complex signals coming from activated G protein-coupled receptors.