SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1-involved EMT in hepatocellular carcinoma.

BACKGROUND: The protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression. METHODS: The modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient's overall survival versus AnxA6 expression level was evaluated by the Kaplan-Meier method. RESULTS: Lys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients. CONCLUSIONS: AnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth.

BACKGROUND: The Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells. METHODS: Cells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC-MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model. RESULTS: AnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells. CONCLUSIONS: Besides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect. Video Abstract.

Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer.

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling-liquid chromatography-tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.

Reciprocal expression of Annexin A6 and RasGRF2 discriminates rapidly growing from invasive triple negative breast cancer subsets.

Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.

Construction of an Immune-Associated Gene-Based Signature in Muscle-Invasive Bladder Cancer.

BACKGROUND: In recent years, immune-associated genes (IAGs) have been documented as having critical roles in the occurrence and progression of muscle-invasive bladder cancer (MIBC). Novel immune-related biomarkers and a robust prognostic signature for MIBC patients are still limited. The study is aimed at developing an IAG-based signature to predict the prognosis of MIBC patients. METHODS: In the present study, we identified differentially expressed IAGs in MIBC by using transcriptomics data from The Cancer Genome Atlas (TCGA) database and proteomics data from our samples. We further constructed an IAG-based signature and evaluated its prognostic and predictive value by survival analysis and nomogram. Tumor Immune Estimation Resource (TIMER) was applied to explore the correlation between the IAG-based signature and immune cell infiltration in the microenvironment of MIBC. RESULTS: A total of 22 differentially expressed IAGs were identified, and 2 IAGs (NR2F6 and AHNAK) were used to establish a prognostic signature. Subsequently, survival analysis showed that high-risk scores were significantly correlated with poor overall survival (OS), progression-free survival (PFS), and disease-free survival (DFS) of MIBC patients. A prognostic nomogram was constructed by integrating clinical factors with the IAG-based signature risk score. In addition, the IAG-based signature risk score was positively associated with the infiltration of macrophages and dendritic cells in MIBC. CONCLUSIONS: We constructed and verified a novel IAG-based signature, which could predict the prognosis of MIBC and might reflect the status of the immune microenvironment of MIBC. Further studies in more independent clinical cohorts and further experimental exploration of the prognostic IAG-based signature are still needed.

Annexin A6 improves anti-migratory and anti-invasive properties of tyrosine kinase inhibitors in EGFR overexpressing human squamous epithelial cells.

Annexin A6 (AnxA6), a member of the calcium (Ca2+ ) and membrane binding annexins, is known to stabilize and establish the formation of multifactorial signaling complexes. At the plasma membrane, AnxA6 is a scaffold for protein kinase Cα (PKCα) and GTPase-activating protein p120GAP to promote downregulation of epidermal growth factor receptor (EGFR) and Ras/mitogen-activated protein kinase (MAPK) signaling. In human squamous A431 epithelial carcinoma cells, which overexpress EGFR, but lack endogenous AnxA6, restoration of AnxA6 expression (A431-A6) promotes PKCα-mediated threonine 654 (T654)-EGFR phosphorylation, which inhibits EGFR tyrosine kinase activity. This is associated with reduced A431-A6 cell growth, but also decreased migration and invasion in wound healing, matrigel, and organotypic matrices. Here, we show that A431-A6 cells display reduced EGFR activity in vivo, with xenograft analysis identifying increased pT654-EGFR levels, but reduced tyrosine EGFR phosphorylation compared to controls. In contrast, PKCα depletion in A431-A6 tumors is associated with strongly reduced pT654 EGFR levels, yet increased EGFR tyrosine phosphorylation and MAPK activity. Moreover, tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib) more effectively inhibit cell viability, clonogenic growth, and wound healing of A431-A6 cells compared to controls. Likewise, the ability of AnxA6 to inhibit A431 motility and invasiveness strongly improves TKI efficacy in matrigel invasion assays. This correlates with a greatly reduced invasion of the surrounding matrix of TKI-treated A431-A6 when cultured in 3D spheroids. Altogether, these findings implicate that elevated AnxA6 scaffold levels contribute to improve TKI-mediated inhibition of growth and migration, but also invasive properties in EGFR overexpressing human squamous epithelial carcinoma.

A siRNA-based method for efficient silencing of PYROXD1 gene expression in the colon cancer cell line HCT116.

PURPOSE: The aim of this study was to determine the biological function of pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1), a recently discovered protein, in colon cancer cell line HCT116. METHODS: The small interfering RNA (siRNA) was designed rationally on the basis of the target sequence against PYROXD1. Relative PYROXD1 mRNA levels were measured by a quantitative real-time polymerase chain reaction. Flow cytometry was performed to monitor tumor cells proliferation and apoptosis after siRNA transfection. RESULTS: Knockdown of PYROXD1 arrested the cell cycle, and induced late apoptosis in colon cancer cell line HCT116 DISCUSSION: Taken together, these results revealed the critical roles of PYROXD1 in regulating cell cycle and apoptosis and possibly will signify its therapeutic potential for targeting colorectal cancer models.

Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.

Annexins A2 and A6 interact with the extreme N terminus of tau and thereby contribute to tau's axonal localization.

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca2+-regulated plasma membrane-binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca2+-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau-annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau-annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.

Annexin A6-A multifunctional scaffold in cell motility.

Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca2+)-dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.

Annexin A6 protein is downregulated in human hepatocellular carcinoma.

Annexin A6 (AnxA6) is a lipid-binding protein highly expressed in the liver, regulating cholesterol homeostasis and signaling pathways with a role in liver physiology. Here, we analyzed whether hepatic AnxA6 levels are affected by pathological conditions that are associated with liver dysfunction and liver injury. AnxA6 levels in the fatty liver of mice fed a high-fat diet, in ob/ob and db/db animals and in human fatty liver are comparable to controls. Similarly, AnxA6 levels appear unaffected in murine nonalcoholic steatohepatitis and human liver fibrosis. Accordingly, adiponectin, lysophosphatidylcholine, palmitate, and TGFbeta, all of which have a role in liver injury, do not affect AnxA6 expression in human hepatocytes. Likewise, adiponectin and IL8 do not alter AnxA6 levels in primary human hepatic stellate cells. However, in hepatic tumors of 18 patients, AnxA6 protein levels are substantially reduced compared to nontumorous tissues. AnxA6 mRNA is even increased in the tumors suggesting that posttranscriptional mechanisms are involved herein. Lipidomic analysis shows trends toward elevated cholesteryl ester and sphingomyelin in the tumor samples, yet the ratio of tumor to nontumorous AnxA6 does not correlate with these lipids. The current study shows that AnxA6 is specifically reduced in human hepatocellular carcinoma suggesting a role of this protein in hepatocarcinogenesis.

GNB2 is a mediator of lidocaine-induced apoptosis in rat pheochromocytoma PC12 cells.

Lidocaine has been recognized to induce neurotoxicity. However, the molecular mechanism underlying this effect, especially the critical molecules in cells that mediated the lidocaine-induced apoptosis were unclear. In the present study, PC12 cells were administrated with lidocaine for 48h. Using MTT assay and flow cytometry, we found lidocaine significantly decreased the cell proliferation and S phases in PC12 cells with treatment concentrations, and significantly enhanced cell apoptosis with treatment concentrations. Two-dimensional gel electrophoresis (2-DE) analysis and LC-MS/MS were used to identification of protein biomarkers. Six proteins were identified. Among them, three were up-expressed including ANXA6, GNB2 and STMN1, other three were down-expressed including ubiquitin-linke protein 7 (UBL7), DDAH2 and BLVRB. Using qRT-PCR, we confirmed that lidocaine up-regulated the mRNA expression of STMN1, GNB2, ANXA6 and DDAH2, and found that the GNB2 had the largest change (about increased by 6.4 folds). The up-regulation of GNB2 by lidocaine was also validated by western blot. After transfected with 100µM GNB2-Rat-453 siRNA, the expression of GNB2 in PC12 cells was almost completely inhibited; and the cell proliferation and cells in S phases were significantly enhanced, cell apoptosis including both early apoptosis and later apoptosis were significantly reduced in the presence of 0.5mM lidocaine for 48h. Therefore, neuronal apoptosis was induced by lidocaine and this effect was mediated by GNB2. Further research is needed to assess the clinical relevance and exact mechanism of neuronal apoptosis caused by lidocaine.

Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration.

Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.

Genome-wide transcriptional profiling analysis reveals annexin A6 as a novel EZH2 target gene involving gastric cellular proliferation.

A histone methyltransferase enhancer of zeste homologue 2 (EZH2) catalyzes trimethylation at histone H3 lysine27 (H3K27me3) and is frequently dysregulated in a wide range of human cancers. EZH2-mediated gene silencing contributes to carcinogenesis and regulates stem cell maintenance and differentiation; however, the underlining mechanisms remain to be completely understood. Here, we found that downregulation of EZH2 by RNA interference (RNAi) in gastric cancer cells suppresses cell growth, migration, invasion, and induces cell cycle arrest. Transcriptome analysis identified 1223 EZH2 responsive genes upon EZH2 knockdown. These genes are involved in the biological processes of cell cycle, proliferation and metastasis. Particularly, we found that annexin A6 (ANXA6) is a new target of EZH2 and is repressed in gastric cancer cells. Restoration of ANXA6 expression inhibits gastric cellular proliferation. We further demonstrated that EZH2-mediated H3K27me3, rather than promoter DNA methylation, is primarily responsible for ANXA6 inhibition. Taken together, our results provide a framework for understanding EZH2 biology and reveal ANXA6 as a new EZH2 target involving gastric cellular proliferation.

Differential skeletal muscle proteome of high- and low-active mice.

Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal.

Reduced annexin A6 expression promotes the degradation of activated epidermal growth factor receptor and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors.

BACKGROUND: The expression of annexin A6 (AnxA6) in AnxA6-deficient non-invasive tumor cells has been shown to terminate epidermal growth factor receptor (EGFR) activation and downstream signaling. However, as a scaffolding protein, AnxA6 may stabilize activated cell-surface receptors to promote cellular processes such as tumor cell motility and invasiveness. In this study, we investigated the contribution of AnxA6 in the activity of EGFR in invasive breast cancer cells and examined whether the expression status of AnxA6 influences the response of these cells to EGFR-targeted tyrosine kinase inhibitors (TKIs) and/or patient survival. RESULTS: We demonstrate that in invasive BT-549 breast cancer cells AnxA6 expression is required for sustained membrane localization of activated (phosho-Y1068) EGFR and consequently, persistent activation of MAP kinase ERK1/2 and phosphoinositide 3-kinase/Akt pathways. Depletion of AnxA6 in these cells was accompanied by rapid degradation of activated EGFR, attenuated downstream signaling and as expected enhanced anchorage-independent growth. Besides inhibition of cell motility and invasiveness, AnxA6-depleted cells were also more sensitive to the EGFR-targeted TKIs lapatinib and PD153035. We also provide evidence suggesting that reduced AnxA6 expression is associated with a better relapse-free survival but poorer distant metastasis-free and overall survival of basal-like breast cancer patients. CONCLUSIONS: Together this demonstrates that the rapid degradation of activated EGFR in AnxA6-depleted invasive tumor cells underlies their sensitivity to EGFR-targeted TKIs and reduced motility. These data also suggest that AnxA6 expression status may be useful for the prediction of the survival and likelihood of basal-like breast cancer patients to respond to EGFR-targeted therapies.

Annexin A6-balanced late endosomal cholesterol controls influenza A replication and propagation.

UNLABELLED: Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca(2+)-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis. IMPORTANCE: Influenza A virus (IAV) is a major public health concern, and yet, major host-pathogen interactions regulating IAV replication still remain poorly understood. It is known that host cell cholesterol is a critical factor in the influenza virus life cycle. The viral envelope is derived from the host cell membrane during the process of budding and, hence, equips the virus with a special lipid-protein mixture which is high in cholesterol. However, the influence of host cell cholesterol homeostasis on IAV infection is largely unknown. We show that IAV infection success critically depends on host cell cholesterol distribution. Cholesterol sequestration in the endosomal compartment impairs progeny titer and infectivity and is associated with reduced cholesterol content in the viral envelope.

Annexin A6 interacts with p65 and stimulates NF-κB activity and catabolic events in articular chondrocytes.

OBJECTIVE: ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. METHODS: Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1ß (IL-1ß) or tumor necrosis factor α (TNFα), and expression of catabolic genes and activation of NF-κB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1ß or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1ß-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-κB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1ß-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. RESULTS: Loss of Anxa6 resulted in decreased NF-κB activation and catabolic marker messenger RNA (mRNA) levels in IL-1ß- or TNFα-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-κB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-κB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1ß injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. CONCLUSION: Our data define a role of annexin A6 in the modulation of NF-κB activity and in the stimulation of catabolic events in articular chondrocytes.