Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

AnnexinA7 promotes epithelial–mesenchymal transition by interacting with Sorcin and contributes to aggressiveness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and metastasis is the major cause of the high mortality of HCC. In this study, we identified that AnnexinA7 (ANXA7) and Sorcin (SRI) are overexpressed and interacting proteins in HCC tissues and cells. In vitro functional investigations revealed that the interaction between ANXA7 and SRI regulated epithelial–mesenchymal transition (EMT), and then affected migration, invasion, and proliferation in HCC cells. Furthermore overexpression/knockdown of ANXA7 was remarkably effective in promoting/inhibiting tumorigenicity and EMT in vivo. Altogether, our study unveiled a mechanism that ANXA7 promotes EMT by interacting with SRI and further contributes to the aggressiveness in HCC, which provides a novel potential therapeutic target for preventing recurrence and metastasis in HCC.Subject terms: Medical research, Genetics research     

LUCAT1 contributes to MYRF‐dependent smooth muscle cell apoptosis and may facilitate aneurysm formation via the sequestration of miR‐199a‐5p

The overwhelming number of interrogations reveals the implication of long noncoding RNAs (lncRNAs) in diverse malignancies, little is unveiled about lncRNAs participation in the abdominal aortic aneurysm (AAA). The study aimed to monitor the role and responsible mechanism of LUCAT1 in AAA. The cellular function of LUCAT1 on smooth muscle cells (SMCs) proliferation and apoptosis were examined through the conduction of CCK‐8, EdU, TUNEL, and caspase‐3 activity assays. LUCAT1 depletion was observed to boost SMCs proliferation or suppress SMCs apoptosis. The opposite results on SMCs proliferation and apoptosis were achieved in response to LUCAT1 promotion. The abundance of LUCAT1 in the cytoplasm was ascertained by subcellular fractionation and FISH analyses on the basis of LncLocator prediction. The binding of LUCAT1 to miR‐199a‐5p predicted by DIANA and starbase was certified by luciferase reporter assay and RIP analysis. Besides, multiple prediction tools unveiled the interaction between miR‐199a‐5p and myelin regulatory factor (MYRF). Quantitative real‐time polymerase chain reaction uncovered the suppressive effect of miR‐199a‐5p and the positive regulation of LUCAT1 on MYRF expression. Rescue experiments revealed that LUCAT1 depletion pose suppression on SMCs apoptosis and MYRF elevation abrogated this suppression induced by LUCAT1 inhibition. These findings unmasked that the pro‐apoptosis impact of LUCAT1 in SMCs via directly targeting miR‐199a‐5p to elevate MYRF expression, which may provide valuable information on AAA prevention.     

Exosomal ANXA2 derived from ovarian cancer cells regulates epithelial-mesenchymal plasticity of human peritoneal mesothelial cells

Ovarian cancer, one of the malignant gynaecological tumours with the highest mortality rate among female reproductive system, is prone to metastasis, recurrence and chemotherapy resistance, causing a poor prognosis. Exosomes can regulate the epithelial-mesenchymal plasticity of tumour cells, remodel surrounding tumour microenvironment, and affect tumour cell proliferation, invasion and metastasis. However, the function and mechanism of exosomes in the intraperitoneal implantation of ovarian cancer remain unclear. In this study, exosomal annexin A2 (ANXA2) derived from ovarian cancer cells was co-cultured with human peritoneal mesothelial (HMrSV5) cells; functional experiments were conducted to explore the effects of exosomal ANXA2 on the biological behaviour of HMrSV5 and the related mechanisms. This study showed that ANXA2 in ovarian cancer cells can be transferred to HMrSV5 cells through exosomes, exosomal ANXA2 can not only promote the migration, invasion and apoptosis of HMrSV5 cells, but also regulates morphological changes and fibrosis of HMrSV5 cells. Furthermore, ANXA2 promotes the mesothelial-mesenchymal transition (MMT) and degradation of the extracellular matrix of HMrSV5 cells through PI3K/AKT/mTOR pathway, finally affects pre-metastasis microenvironment of ovarian cancer, which provides a new theoretical basis for the mechanism of intraperitoneal implantation and metastasis of ovarian cancer.     

Cancer-associated fibroblasts promote epithelial-mesenchymal transition and EGFR-TKI resistance of non-small cell lung cancers via HGF/IGF-1/ANXA2 signaling

The involvement of the tumor stromal cells in acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism remains unclear. In the present study, we investigated the role and mechanism underlying Cancer-associated fibroblasts (CAFs) in TKI resistance of NSCLCs. In vitro and in vivo experiments showed that HCC827 and PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR-TKI gefitinib when cultured with CAFs isolated from NSCLC tissues. Moreover, we showed that CAFs could induce epithelial-mesenchymal transition (EMT) phenotype of HCC827 and PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition markers. Using proteomics-based method, we identified that CAFs significantly increased the expression of the Annexin A2 (ANXA2). More importantly, knockdown of ANXA2 completely reversed EMT phenotype and gefitinib resistance induced by CAFs. Furthermore, we found that CAFs increased the expression and phosphorylation of ANXA2 by secretion of growth factors HGF and IGF-1 and by activation of the corresponding receptors c-met and IGF-1R. Dual inhibition of HGF/c-met and IGF-1/IGF-1R pathways could significantly suppress ANXA2, and markedly reduced CAFs-induced EMT and gefitinib resistance. Taken together, these findings indicate that CAFs promote EGFR-TKIs resistance through HGF/IGF-1/ANXA2/EMT signaling and may be an ideal therapeutic target in NSCLCs with EGFR-activating mutations.     

Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

High mobility group protein box1 (HMGB1) and its receptor—receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.     

SRXN1 stimulates hepatocellular carcinoma tumorigenesis and metastasis through modulating ROS/p65/BTG2 signalling

Sulfiredoxin 1 (SRXN1) is a pivotal regulator of the antioxidant response in eukaryotic cells. However, the role of SRXN1 in hepatocellular carcinoma (HCC) is far from clear. The present study aims to elucidate whether SRXN1 participates in tumorigenesis and metastasis of HCC and to determine the molecular mechanisms. We found that SRXN1 expression was up‐regulated in HCC tissue samples and correlated with poor prognosis in HCC patients. We also observed that SRXN1 knockdown by transient siRNA transfection inhibited HCC cell proliferation, migration and invasion. Overexpression of SRXN1 increased HCC cell migration and invasion. B‐cell translocation gene 2 (BTG2) was identified as a downstream target of SRXN1. Mechanistic studies revealed that SRXN1‐depleted reactive oxygen species (ROS) modulated migration and invasion of HCC cells. In addition, the ROS/p65/BTG2 signalling hub was found to regulate the epithelial‐mesenchymal transition (EMT), which mediates the pro‐metastasis role of SRXN1 in HCC cells. In vivo experiments showed SRXN1 promotes HCC tumour growth and metastasis in mouse subcutaneous xenograft and metastasis models. Collectively, our results revealed a novel pro‐tumorigenic and pro‐metastatic function of SRXN1 in HCC. These findings demonstrate a rationale to exploit SRXN1 as a therapeutic target effectively preventing metastasis of HCC.     

Long non‐coding RNA SNAI3‐AS1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the UPF1/Smad7 signalling pathway

Emerging evidence has indicated that deregulation of long non‐coding RNAs (lncRNAs) can contribute to the progression of human cancers, including hepatocellular carcinoma (HCC). However, the role and exact mechanism of most lncRNAs in tumours remains largely unknown. In the current study, we found a novel long non‐coding RNA termed SNAI3‐AS1 which was generally up‐regulated in HCC tissues compared with normal control. Higher expression of SNAI3‐AS1 was significantly correlated with shorter overall survival of HCC patients. Knockdown of SNAI3‐AS1 inhibited the proliferation and metastasis of HCC cells in vitro, whereas overexpression of SNAI3‐AS1 promoted the proliferation and metastasis of HCC cells. Further investigations showed that SNAI3‐AS1 could affect HCC tumorigenesis by binding up‐frameshift protein 1 (UPF1), regulating Smad7 expression and activating TGF‐β/Smad pathway. Functionally, SNAI3‐AS1 promoted HCC growth and metastasis by inducing tumour epithelial to mesenchymal transition (EMT). Taken together, these findings showed that SNAI3‐AS1 promotes the progression of HCC by regulating the UPF1 and activating TGF‐β/Smad pathway.     

Long non‐coding RNA deleted in lymphocytic leukaemia 1 promotes hepatocellular carcinoma progression by sponging miR‐133a to regulate IGF‐1R expression

Long non‐coding RNA (lncRNA) deleted in lymphocytic leukaemia 1 (DLEU1) was reported to be involved in the occurrence and development of multiple cancers. However, the exact expression, biological function and underlying mechanism of DLEU1 in hepatocellular carcinoma (HCC) remain unclear. In this study, real‐time quantitative polymerase chain reaction (qRT‐PCR) in HCC tissues and cell lines revealed that DLEU1 expression was up‐regulated, and the increased DLEU1 was closely associated with advanced tumour‐node‐metastasis stage, vascular metastasis and poor overall survival. Function experiments showed that knockdown of DLEU1 significantly inhibited HCC cell proliferation, colony formation, migration and invasion, and suppressed epithelial to mesenchymal transition (EMT) process via increasing the expression of E‐cadherin and decreasing the expression of N‐cadherin and Vimentin. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay demonstrated that DLEU1 could sponge miR‐133a. Moreover, miR‐133a inhibition significantly reversed the suppression effects of DLEU1 knockdown on HCC cells. Besides, we found that silenced DLEU1 significantly decreased insulin‐like growth factor 1 receptor (IGF‐1R) expression (a target of miR‐133a) and its downstream signal PI3K/AKT pathway in HCC cells, while miR‐133a inhibitor partially reversed this trend. Furthermore, DLEU1 knockdown impaired tumour growth in vivo by regulating miR‐133a/IGF‐1R axis. Collectively, these findings indicate that DLEU1 promoted HCC progression by sponging miR‐133a to regulate IGF‐1R expression. Deleted in lymphocytic leukaemia 1/miR‐133a/IGF‐1R axis may be a novel target for treatment of HCC.     

The role of EGF‐EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF‐EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio‐behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ‐235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF‐EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross‐talk between CXCR2 and EGFR. EGF‐EGFR signaling pathway can be the potential target of therapies for HCC.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

LncRNA FOXD2‐AS1 as a competitive endogenous RNA against miR‐150‐5p reverses resistance to sorafenib in hepatocellular carcinoma

The current study elucidated the role of a long non‐coding RNA (lncRNA), FOXD2‐AS1, in the pathogenesis of hepatocellular carcinoma (HCC) and the regulatory mechanism underlying FOXD2‐AS1/miR‐150‐5p/transmembrane protein 9 (TMEM9) signalling in HCC. Microarray analysis was used for preliminary screening of candidate lncRNAs in HCC tissues. qRT‐PCR and Western blot analyses were used to detect the expression of FOXD2‐AS1. Cell proliferation assays, luciferase assay and RNA immunoprecipitation were performed to examine the mechanism by which FOXD2‐AS1 mediates sorafenib resistance in HCC cells. FOXD2‐AS1 and TMEM9 were significantly decreased and miR‐150‐5p was increased in SR‐HepG2 and SR‐HUH7 cells compared with control parental cells. Overexpression of FOXD2‐AS1 increased TMEM9 expression and overcame the resistance of SR‐HepG2 and SR‐HUH7 cells. Conversely, knockdown of FOXD2‐AS1 decreased TMEM9 expression and increased the sensitivity of HepG2 and Huh7 cells to sorafenib. Our data also demonstrated that FOXD2‐AS1 functioned as a sponge for miR‐150‐5p to modulate TMEM9 expression. Taken together, our findings revealed that FOXD2‐AS1 is an important regulator of TMEM9 and contributed to sorafenib resistance. Thus, FOXD2‐AS1 may serve as a therapeutic target against sorafenib resistance in HCC.     

Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca²⁺-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca²⁺ perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca²⁺. These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.