Exosomes: the key of sophisticated cell–cell communication and targeted metastasis in pancreatic cancer

Pancreatic cancer is one of the most common malignancies. Unfortunately, the lack of effective methods of treatment and diagnosis has led to poor prognosis coupled with a very high mortality rate. So far, the pathogenesis and progression mechanisms of pancreatic cancer have been poorly characterized. Exosomes are small vesicles secreted by most cells, contain lipids, proteins, and nucleic acids, and are involved in diverse functions such as intercellular communications, biological processes, and cell signaling. In pancreatic cancer, exosomes are enriched with multiple signaling molecules that mediate intercellular communication with control of immune suppression, mutual promotion between pancreas stellate cells and pancreatic cancer cells, and reprogramming of normal cells. In addition, exosomes can regulate the pancreatic cancer microenvironment and promote the growth and survival of pancreatic cancer. Exosomes can also build pre-metastatic micro-ecological niches and facilitate the targeting of pancreatic cancer. The ability of exosomes to load cargo and target allows them to be of great clinical value as a biomarker mediator for targeted drugs in pancreatic cancer.

Na+/Ca2+ exchanger isoform 1 takes part to the Ca2+-related prosurvival pathway of SOD1 in primary motor neurons exposed to beta-methylamino-l-alanine

Background The cycad neurotoxin beta-methylamino-l-alanine (L-BMAA), one of the environmental trigger factor for amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC), may cause neurodegeneration by disrupting organellar Ca2+ homeostasis. Through the activation of Akt/ERK1/2 pathway, the Cu,Zn-superoxide dismutase (SOD1) and its non-metallated form, ApoSOD1, prevent endoplasmic reticulum (ER) stress-induced cell death in motor neurons exposed to L-BMAA. This occurs through the rapid increase of intracellular Ca2+ concentration ([Ca2+]i) in part flowing from the extracellular compartment and in part released from ER. However, the molecular components of this mechanism remain uncharacterized. Methods By an integrated approach consisting on the use of siRNA strategy, Western blotting, confocal double- labeling immunofluorescence, patch-clamp electrophysiology, and Fura 2-/SBFI-single-cell imaging, we explored in rat motor neuron-enriched cultures the involvement of the plasma membrane proteins Na+/Ca2+ exchanger (NCX) and purinergic P2X7 receptor as well as that of the intracellular cADP-ribose (cADPR) pathway, in the neuroprotective mechanism of SOD1. Results We showed that SOD1-induced [Ca2+]i rise was prevented neither by A430879, a P2X7 receptor specific antagonist or 8-bromo-cADPR, a cell permeant antagonist of cADP-ribose, but only by the pan inhibitor of NCX, CB-DMB. The same occurred for the ApoSOD1. Confocal double labeling immunofluorescence showed a huge expression of plasmalemmal NCX1 and intracellular NCX3 isoforms. Furthermore, we identified NCX1 reverse mode as the main mechanism responsible for the neuroprotective ER Ca2+ refilling elicited by SOD1 and ApoSOD1 through which they promoted translocation of active Akt in the nuclei of a subset of primary motor neurons. Finally, the activation of NCX1 by the specific agonist CN-PYB2 protected motor neurons from L-BMAA-induced cell death, mimicking the effect of SOD1. Conclusion Collectively, our data indicate that SOD1 and ApoSOD1 exert their neuroprotective effect by modulating ER Ca2+ content through the activation of NCX1 reverse mode and Akt nuclear translocation in a subset of primary motor neurons.

Extracellular vesicles derived from glioblastoma promote proliferation and migration of neural progenitor cells via PI3K-Akt pathway

Background Glioblastomas are lethal brain tumors under the current combinatorial therapeutic strategy that includes surgery, chemo- and radio-therapies. Extensive changes in the tumor microenvironment is a key reason for resistance to chemo- or radio-therapy and frequent tumor recurrences. Understanding the tumor-nontumor cell interaction in TME is critical for developing new therapy. Glioblastomas are known to recruit normal cells in their environs to sustain growth and encroachment into other regions. Neural progenitor cells (NPCs) have been noted to migrate towards the site of glioblastomas, however, the detailed mechanisms underlying glioblastoma-mediated NPCs’ alteration remain unkown. Methods We collected EVs in the culture medium of three classic glioblastoma cell lines, U87 and A172 (male cell lines), and LN229 (female cell line). U87, A172, and LN229 were co-cultured with their corresponding EVs, respectively. Mouse NPCs (mNPCs) were co-cultured with glioblastoma-derived EVs. The proliferation and migration of tumor cells and mNPCs after EVs treatment were examined. Proteomic analysis and western blotting were utilized to identify the underlying mechanisms of glioblastoma-derived EVs-induced alterations in mNPCs. Results We first show that glioblastoma cell lines U87-, A172-, and LN229-derived EVs were essential for glioblastoma cell prolifeartion and migration. We then demonstrated that glioblastoma-derived EVs dramatically promoted NPC proliferation and migration. Mechanistic studies identify that glioblastoma-derived EVs achieve their functions via activating PI3K-Akt-mTOR pathway in mNPCs. Inhibiting PI3K-Akt pathway reversed the elevated prolfieration and migration of glioblastoma-derived EVs-treated mNPCs. Conclusion Our findings demonstrate that EVs play a key role in intercellular communication in tumor microenvironment. Inhibition of the tumorgenic EVs-mediated PI3K-Akt-mTOR pathway activation might be a novel strategy to shed light on glioblastoma therapy.

Comprehensive analysis of a TNF family based-signature in diffuse gliomas with regard to prognosis and immune significance

Background Several studies have shown that members of the tumor necrosis factor (TNF) family play an important role in cancer immunoregulation, and trials targeting these molecules are already underway. Our study aimed to integrate and analyze the expression patterns and clinical significance of TNF family-related genes in gliomas. Methods A total of 1749 gliomas from 4 datasets were enrolled in our study, including the Cancer Genome Atlas (TCGA) dataset as the training cohort and the other three datasets (CGGA, GSE16011, and Rembrandt) as validation cohorts. Clinical information, RNA expression data, and genomic profile were collected for analysis. We screened the signature gene set by Cox proportional hazards modelling. We evaluated the prognostic value of the signature by Kaplan–Meier analysis and timeROC curve. Gene Ontology (GO) and Gene set enrichment analysis (GSEA) analysis were performed for functional annotation. CIBERSORT algorithm and inflammatory metagenes were used to reveal immune characteristics. Results In gliomas, the expression of most TNF family members was positively correlated. Univariate analysis showed that most TNF family members were related to the overall survival of patients. Then through the LASSO regression model, we developed a TNF family-based signature, which was related to clinical, molecular, and genetic characteristics of patients with glioma. Moreover, the signature was found to be an independent prognostic marker through survival curve analysis and Cox regression analysis. Furthermore, a nomogram prognostic model was constructed to predict individual survival rates at 1, 3 and 5 years. Functional annotation analysis revealed that the immune and inflammatory response pathways were enriched in the high-risk group. Immunological analysis showed the immunosuppressive status in the high-risk group. Conclusions We developed a TNF family-based signature to predict the prognosis of patients with glioma.

Functional signaling test identifies HER2 negative breast cancer patients who may benefit from c-Met and pan-HER combination therapy

Background Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions. Methods In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient’s ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies. Results The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients’ cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies. Conclusions The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy.

GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch

Background G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. Methods Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein–protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. Results We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. Conclusion Our results show a stimulated shift of PP2Ac from PI3K to AKT termed “PP2A switch” that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation.

Scavenger receptors in host defense: from functional aspects to mode of action

Scavenger receptors belong to a superfamily of proteins that are structurally heterogeneous and encompass the miscellaneous group of transmembrane proteins and soluble secretory extracellular domain. They are functionally diverse as they are involved in various disorders and biological pathways and their major function in innate immunity and homeostasis. Numerous scavenger receptors have been discovered so far and are apportioned in various classes (A-L). Scavenger receptors are documented as pattern recognition receptors and known to act in coordination with other co-receptors such as Toll-like receptors in generating the immune responses against a repertoire of ligands such as microbial pathogens, non-self, intracellular and modified self-molecules through various diverse mechanisms like adhesion, endocytosis and phagocytosis etc. Unlike, most of the scavenger receptors discussed below have both membrane and soluble forms that participate in scavenging; the role of a potential scavenging receptor Angiotensin-Converting Enzyme-2 has also been discussed whereby only its soluble form might participate in preventing the pathogen entry and replication, unlike its membrane-bound form. This review majorly gives an insight on the functional aspect of scavenger receptors in host defence and describes their mode of action extensively in various immune pathways involved with each receptor type.

Small extracellular vesicles: from mediating cancer cell metastasis to therapeutic value in pancreatic cancer

Pancreatic cancer is a highly malignant tumor and, is extremely difficult to diagnose and treat. Metastasis is one of the critical steps in the development of cancer and uses cell to cell communication to mediate changes in the microenvironment. Small extracellular vesicles (sEVs)-carry proteins, nucleic acids and other bioactive substances, and are important medium for communication between cells. There are two primary steps in sVEs-mediated metastasis: communication between pancreatic cancer cells and their surrounding microenvironment; and the communication between primary tumor cells and distant organ cells in distant organs that promotes angiogenesis, reshaping extracellular matrix, forming immunosuppressive environment and other ways to form appropriate pre-metastasis niche. Here, we explore the mechanism of localization and metastasis of pancreatic cancer and use sEVs as early biomarkers for the detection and treatment of pancreatic cancer. Graphical Abstract

CBX4-dependent regulation of HDAC3 nuclear translocation reduces Bmp2-induced osteoblastic differentiation and calcification in adamantinomatous craniopharyngioma

Background Calcification of adamantinomatous craniopharyngioma (ACP) often causes problems with tumor resection, leading to a high incidence of deadly complications and tumor recurrence. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are 2 key enzymes that regulate histone acetylation and play important roles in tumor development. However, the roles of HAT and HDAC in the calcification and osteoblastic differentiation of ACP are not known. Methods In this study, primary cells were isolated from ACP tissues, and calcification was induced with bone morphogenetic protein 2 (Bmp2). HDAC3 expression was assessed in 12 tissue samples by Western blotting and immunohistochemistry. ACP calcification was assessed by Alizarin red staining. A luciferase reporter assay was performed to examine the interaction between miR-181b and the 3’-untranslated region of the polycomb chromobox 4 (CBX4) gene. Results Our results showed that the expression of HDAC3 was increased in the calcified ACP samples, but inhibition of HDAC3 promoted ACP cell calcification and osteoblastic differentiation. Mechanistically, HDAC3 nuclear translocation was suppressed by Bmp2, leading to Runx2 protein expression and Osterix, osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) mRNA expression. In addition, this process was suppressed by CBX4, which stabilized the nuclear localization of HDAC3. miR-181b, the expression of which was increased in Bmp2-induced ACP cells, directly targeted and decreased CBX4 expression and inhibited the nuclear localization of HDAC3. Conclusions Our results demonstrate that Bmp2 increases miR-181b levels to directly target and inhibit CBX4 expression, leading to a reduction in the CBX4-dependent regulation of HDAC3 nuclear translocation, which results in Runx2 activation/osteoblastic differentiation and calcium deposition in ACP. Further studies targeting these cascades may contribute to therapeutic interventions used for recurrent ACP.

Cytoplasmic eIF6 promotes OSCC malignant behavior through AKT pathway

Background Eukaryotic translation initiation factor 6 (eIF6), also known as integrin β4 binding protein, is involved in ribosome formation and mRNA translation, acting as an anti-association factor. It is also essential for the growth and reproduction of cells, including tumor cells. Yet, its role in oral squamous cell carcinoma (OSCC) remains unclear. Methods The expression characteristics of eIF6 in 233 samples were comprehensively analyzed by immunohistochemical staining (IHC). Effects of eIF6 over-expression and knockdown on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Western blot, immunofluorescence (IF) and co-immunoprecipitation (co-IP) were performed for mechanical verification. Results We found that cytoplasmic eIF6 was abnormally highly expressed in OSCC tissues, and its expression was associated with tumor size and the clinical grade. Amplification of eIF6 promoted the growth, migration and invasion capabilities of OSCC cell lines in vitro and tumor growth in vivo. Through Western blot analysis, we further discovered that eIF6 significantly promotes epithelial-mesenchymal transformation (EMT) in OSCC cells, while depletion of eIF6 can reverse this process. Mechanistically, eIF6 promoted tumor progression by activating the AKT signaling pathway. By performing co-immunoprecipitation, we discovered a direct interaction between endogenous eIF6 and AKT protein in the cytoplasm. Conclusion These results demonstrated that eIF6 could be a new therapeutic target in OSCC, thus providing a new basis for the prognosis of OSCC patients in the future.