Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.     

Integrin Alpha v Beta 6 (αvβ6) and Its Implications in Cancer Treatment

Integrins are necessary for cell adhesion, migration, and positioning. Essential for inducing signalling events for cell survival, proliferation, and differentiation, they also trigger a variety of signal transduction pathways involved in mediating invasion, metastasis, and squamous-cell carcinoma. Several recent studies have demonstrated that the up- and down-regulation of the expression of αv and other integrins can be a potent marker of malignant diseases and patient prognosis. This review focuses on an arginine-glycine-aspartic acid (RGD)-dependent integrin αVβ6, its biology, and its role in healthy humans. We examine the implications of αVβ6 in cancer progression and the promotion of epithelial-mesenchymal transition (EMT) by contributing to the activation of transforming growth factor beta TGF-β. Although αvβ6 is crucial for proper function in healthy people, it has also been validated as a target for cancer treatment. This review briefly considers aspects of targeting αVβ6 in the clinic via different therapeutic modalities.     

Exogenous Integrin αIIbβ3 Inhibitors Revisited: Past,Present and Future Applications

The integrin αIIbβ3 is the most abundant integrin on platelets. Upon platelet activation, the integrin changes its conformation (inside-out signalling) and outside-in signalling takes place leading to platelet spreading, platelet aggregation and thrombus formation. Bloodsucking parasites such as mosquitoes, leeches and ticks express anticoagulant and antiplatelet proteins, which represent major sources of lead compounds for the development of useful therapeutic agents for the treatment of haemostatic disorders or cardiovascular diseases. In addition to hematophagous parasites, snakes also possess anticoagulant and antiplatelet proteins in their salivary glands. Two snake venom proteins have been developed into two antiplatelet drugs that are currently used in the clinic. The group of proteins discussed in this review are disintegrins, low molecular weight integrin-binding cysteine-rich proteins, found in snakes, ticks, leeches, worms and horseflies. Finally, we highlight various oral antagonists, which have been tested in clinical trials but were discontinued due to an increase in mortality. No new αIIbβ3 inhibitors are developed since the approval of current platelet antagonists, and structure-function analysis of exogenous disintegrins could help find platelet antagonists with fewer adverse side effects.     

Secreted HSP90α-LRP1 Signaling Promotes Tumor Metastasis and Chemoresistance in Pancreatic Cancer

The extracellular heat shock protein 90α (eHSP90α) has been reported to promote cancer cell motility. However, whether pancreatic cancer (PC) cells expressed membrane-bound or secreted HSP90α, as well as its underlying mechanism for PC progression, were still unclear. Our study demonstrated that the amounts of secreted HSP90α proteins were discrepant in multiple PC cells. In addition, highly invasive Capan-2 cells have a higher level of secreted HSP90α compared with those of less invasive PL45 cells. The conditioned medium of Capan-2 cells or recombinant HSP90α treatment stimulated the migration and invasion of PC cells, which could be prevented with a neutralizing anti-HSP90α antibody. Furthermore, secreted HSP90α promoted elements of epithelial–mesenchymal transition in PL45 cells, including increases in vimentin and Snail expressions, decreases in E-cadherin expression, and changes in cell shape towards a mesenchymal phenotype, but these phenomena were reversed by the anti-HSP90α antibody in Capan-2 cells. In addition, high levels of low-density lipoprotein receptor-related protein 1 (LRP1) were associated with worsened patient survival in pancreatic adenocarcinoma. We demonstrated LRP1 as a receptor of eHSP90α for its stimulatory role in metastasis, by activating the AKT pathway. In addition, silencing LRP1 enhanced the chemosensitivity to gemcitabine and doxorubicin in Capan-2 cells. Therefore, our study indicated that blocking secreted HSP90α underlies an aspect of metastasis and chemoresistance in PC.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

TGF-β Signaling: From Tissue Fibrosis to Tumor Microenvironment

Transforming growth factor-β (TGF-β) signaling triggers diverse biological actions in inflammatory diseases. In tissue fibrosis, it acts as a key pathogenic regulator for promoting immunoregulation via controlling the activation, proliferation, and apoptosis of immunocytes. In cancer, it plays a critical role in tumor microenvironment (TME) for accelerating invasion, metastasis, angiogenesis, and immunosuppression. Increasing evidence suggest a pleiotropic nature of TGF-β signaling as a critical pathway for generating fibrotic TME, which contains numerous cancer-associated fibroblasts (CAFs), extracellular matrix proteins, and remodeling enzymes. Its pathogenic roles and working mechanisms in tumorigenesis are still largely unclear. Importantly, recent studies successfully demonstrated the clinical implications of fibrotic TME in cancer. This review systematically summarized the latest updates and discoveries of TGF-β signaling in the fibrotic TME.     

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.     

Inhibition of β-Catenin Activity Abolishes LKB1 Loss-Driven Pancreatic Cystadenoma in Mice

Pancreatic cancer (PC) is the seventh leading cause of cancer death worldwide, and remains one of our most recalcitrant and dismal diseases. In contrast to many other malignancies, there has not been a significant improvement in patient survival over the past decade. Despite advances in our understanding of the genetic alterations associated with this disease, an incomplete understanding of the underlying biology and lack of suitable animal models have hampered efforts to develop more effective therapies. LKB1 is a tumor suppressor that functions as a primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK), which is an important mediator in the regulation of cell growth and epithelial polarity pathways. LKB1 is mutated in a significant number of Peutz–Jeghers syndrome (PJS) patients and in a small proportion of sporadic cancers, including PC; however, little is known about how LKB1 loss contributes to PC development. Here, we report that a reduction in Wnt/β-catenin activity is associated with LKB1 tumor-suppressive properties in PC. Remarkably, in vivo functional analyses of β-catenin in the Pdx-1-Cre LKB1^L/L β-catenin^L/L mouse model compared to LKB1 loss-driven cystadenoma demonstrate that the loss of β-catenin impairs cystadenoma development in the pancreas of Pdx-1Cre LKB1^L/L mice and dramatically restores the normal development and functions of the pancreas. This study further determined the in vivo and in vitro therapeutic efficacy of the β-catenin inhibitor FH535 in suppressing LKB1 loss-driven cystadenoma and reducing PC progression that delineates the potential roles of Wnt/β-catenin signaling in PC harboring LKB1 deficiency.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

Glycogen Synthase Kinase 3β: A True Foe in Pancreatic Cancer

Glycogen synthase kinase 3 beta (GSK-3β) is a serine/threonine protein kinase involved in multiple normal and pathological cell functions, including cell signalling and metabolism. GSK-3β is highly expressed in the onset and progression of multiple cancers with strong involvement in the regulation of proliferation, apoptosis, and chemoresistance. Multiple studies showed pro- and anti-cancer roles of GSK-3β creating confusion about the benefit of targeting GSK-3β for treating cancer. In this mini-review, we focus on the role of GSK-3β in pancreatic cancer. We demonstrate that the proposed anti-cancer roles of GSK-3β are not relevant to pancreatic cancer, and we argue why GSK-3β is, indeed, a very promising therapeutic target in pancreatic cancer.     

Role of Human Primary Renal Fibroblast in TGF-β1-Mediated Fibrosis-Mimicking Devices

Renal fibrosis is a progressive chronic kidney disease that ultimately leads to end-stage renal failure. Despite several approaches to combat renal fibrosis, an experimental model to evaluate currently available drugs is not ideal. We developed fibrosis-mimicking models using three-dimensional (3D) co-culture devices designed with three separate layers of tubule interstitium, namely, epithelial, fibroblastic, and endothelial layers. We introduced human renal proximal tubular epithelial cells (HK-2), human umbilical-vein endothelial cells, and patient-derived renal fibroblasts, and evaluated the effects of transforming growth factor-β (TGF-β) and TGF-β inhibitor treatment on this renal fibrosis model. The expression of the fibrosis marker alpha smooth muscle actin upon TGF-β1 treatment was augmented in monolayer-cultured HK-2 cells in a 3D disease model. In the vascular compartment of renal fibrosis models, the density of vessels was increased and decreased in the TGF-β-treated group and TGF-β-inhibitor treatment group, respectively. Multiplex ELISA using supernatants in the TGF-β-stimulating 3D models showed that pro-inflammatory cytokine and growth factor levels including interleukin-1 beta, tumor necrosis factor alpha, basic fibroblast growth factor, and TGF-β1, TGF-β2, and TGF-β3 were increased, which mimicked the fibrotic microenvironments of human kidneys. This study may enable the construction of a human renal fibrosis-mimicking device model beyond traditional culture experiments.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

αV Integrin Expression and Localization in Male Germ Cells

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.     

Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer

Early detection and discovery of new therapeutic targets are urgently needed to improve the breast cancer treatment outcome. Here we conducted an official clinical trial with cross-validation to corroborate human plasma Hsp90α as a novel breast cancer biomarker. Importantly, similar results were noticed in detecting early-stage breast cancer patients. Additionally, levels of plasma Hsp90α in breast cancer patients were gradually elevated as their clinical stages of regional lymph nodes advanced. In orthotopic breast cancer mouse models, administrating with recombinant Hsp90α protein increased both the primary tumor lymphatic vessel density and sentinel lymph node metastasis by 2 and 10 times, respectively. What is more, Hsp90α neutralizing antibody treatment approximately reduced 70% of lymphatic vessel density and 90% of sentinel lymph node metastasis. In the in vitro study, we demonstrated the role of extracellular Hsp90α (eHsp90α) as a pro-lymphangiogenic factor, which significantly enhanced migration and tube formation abilities of lymphatic endothelial cells (LECs). Mechanistically, eHsp90α signaled to the AKT pathway through low-density lipoprotein receptor-related protein 1 (LRP1) to upregulate the expression and secretion of CXCL8 in the lymphangiogenic process. Collectively, this study proves that plasma Hsp90α serves as an auxiliary diagnosis biomarker and eHsp90α as a molecular mediator promoting lymphangiogenesis in breast cancer.     

Protein Kinase C (Pkc)-δ Mediates Arginine-Induced Glucagon Secretion in Pancreatic α-Cells

The pathophysiology of type 2 diabetes involves insulin and glucagon. Protein kinase C (Pkc)-δ, a serine–threonine kinase, is ubiquitously expressed and involved in regulating cell death and proliferation. However, the role of Pkcδ in regulating glucagon secretion in pancreatic α-cells remains unclear. Therefore, this study aimed to elucidate the physiological role of Pkcδ in glucagon secretion from pancreatic α-cells. Glucagon secretions were investigated in Pkcδ-knockdown InR1G9 cells and pancreatic α-cell-specific Pkcδ-knockout (αPkcδKO) mice. Knockdown of Pkcδ in the glucagon-secreting cell line InR1G9 cells reduced glucagon secretion. The basic amino acid arginine enhances glucagon secretion via voltage-dependent calcium channels (VDCC). Furthermore, we showed that arginine increased Pkcδ phosphorylation at Thr⁵⁰⁵, which is critical for Pkcδ activation. Interestingly, the knockdown of Pkcδ in InR1G9 cells reduced arginine-induced glucagon secretion. Moreover, arginine-induced glucagon secretions were decreased in αPkcδKO mice and islets from αPkcδKO mice. Pkcδ is essential for arginine-induced glucagon secretion in pancreatic α-cells. Therefore, this study may contribute to the elucidation of the molecular mechanism of amino acid-induced glucagon secretion and the development of novel antidiabetic drugs targeting Pkcδ and glucagon.