Cytoskeleton-associated Protein 4 Research Articles

Plasmalemma vesicle-associated protein promotes angiogenesis in cholangiocarcinoma via the DKK1/CKAP4/PI3K signaling pathway.

Cholangiocarcinoma (CCA) is aggressive and has poor clinical outcomes because of typically delayed diagnosis and a lack of effective non-surgical therapeutic options. Recent studies have shown that plasmalemma vesicle-associated protein (PLVAP) is related to angiogenesis in various tumors, and in vivo PLVAP targeting therapy has been proven effective against hepatocellular carcinoma and pancreatic cancer. The goal of this study was to determine the potential therapeutic utility of targeting PLVAP and thus angiogenesis in CCA and explore the underlying molecular mechanisms. We found that the PLVAP expression levels were significantly higher in CCA tissues when compared with matched adjacent non-tumor tissues obtained from a total of 90 CCA patients; higher expression levels of PLVAP were associated with shorter overall survival of patients. In addition, overexpression of PLVAP was associated with higher micro-vessel density in CCA tissues. In a PLVAP overexpressing CCA patient-derived xenograft model, a novel humanized anti-PLVAP antibody in combination with Gemcitabine plus Cisplatin was significantly inhibited tumor growth. Molecular analysis of CCA cells co-cultured with human umbilical vascular endothelial cells or human hepatic sinusoidal endothelial cells showed that Dickkopf-related protein 1 (DKK1) secreted by CCA cells activated the PI3K/Akt pathway after binding to its receptor, cytoskeleton-associated protein 4 (CKAP4), resulting in the upregulation of PLVAP. Thus, CCA cells increased the angiogenic potency of endothelial cells in a paracrine fashion. Consistently, patients bearing CKAP4 and PLVAP overexpressing tumors had a poor prognosis. In conclusion, the DKK1/CKAP4/PI3K/PLVAP pathway increases angiogenesis in CCA and is therefore a potential anti-angiogenic target.

Single-cell RNA Sequencing Analysis Identifies Key Genes in Brain Metastasis from Lung Adenocarcinoma.

Lung adenocarcinoma (LADC) is the most common type of lung cancer and is a subtype of non-small-cell lung cancer (NSCLC). Approximately 40% of LADC patients experience brain metastases (BMs) during the course of the disease. In this study, integrated bioinformatics methods were applied to identify key genes related to brain metastasis in lung adenocarcinoma. We derived and characterized genes differentially expressed between the primary tumour and brain metastases using tumour cells isolated from two lung cancer Patient-derived xenografts (PDX) cases (GSE 69405). Gene ontology (GO) and KEGG pathway enrichment analyses were applied, and protein-protein interaction (PPI) networks and Cytoscape software were utilized to identify key genes. Four key genes, including CKAP4 (Cytoskeleton Associated Protein 4), SERPINA1 (Serpin Family A Member 1), SDC2 (Syndecan 2) and GNG11 (G Protein Subunit Gamma 11) were identified for BM-LADC by the Venn diagram. We believe these key genes may be potential biomarkers for improved prognosis and treatment of lung adenocarcinoma.

Cytoskeleton-Associated Protein 4, a Promising Biomarker for Tumor Diagnosis and Therapy.

Cytoskeleton-associated protein 4 (CKAP4) is located in the rough endoplasmic reticulum (ER) and plays an important role in stabilizing the structure of ER. Meanwhile, CKAP4 is also found to act as an activated receptor at the cell surface. The multifunction of CKAP4 was gradually discovered with growing research evidence. In addition to the involvement in various physiological events including cell proliferation, cell migration, and stabilizing the structure of ER, CKAP4 has been implicated in tumorigenesis. However, the role of CKAP4 is still controversial in tumor biology, which may be related to different signal transduction pathways mediated by binding to different ligands in various microenvironments. Interestingly, CKAP4 has been recently recognized as a serological marker of several tumors and CKAP4 is expected to be a tumor therapeutic target. Therefore, deciphering the gene status, expression regulation, functions of CKAP4 in different diseases may shed new light on CKAP4-based cancer diagnosis and therapeutic strategy. This review discusses the publications that describe CKAP4 in various diseases, especially on tumor promotion and suppression, and provides a detailed discussion on the discrepancy.

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites.

Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.

Detecting Lung Cancer Stages Earlier By Appropriate Markers Rather Than Biopsy And Other Techniques

Abstract Introduction/Objective Early detection of lung cancer stages is deeply under consideration of scientists. By day, high rate of mortality might be caused by cancer of lung over the world. The biopsy is still the golden standard as a histological predictor to determine lung cancer worldwide. However, a biopsy has various complications and hardness especially in collecting tissue samples. Therefore, detecting appropriate tools to determine the early stages of lung cancer is urgently needed. Some biomarkers levels have been determined in patient’s serum. The correlation between studied biomarkers and lung cancer has been studied by current research to avoid biopsy and other techniques. Methods The various biomarkers such as (CEA, SCCA, CYFRA21-1, ProGRP, Prolactin and CKAP4) were measured and calculated in 379 infected cases with lung cancer. Also, the same parameters were determined in 113 healthy persons as controls. Alternative markers’ levels were determined by using ELISA technique. Noninvasive markers were approved by comparison with CT chest scan and Biopsy techniques. Results Our research has indicated that Some of the studied biomarkers such as cytoskeleton-associated protein 4 (CKAP4), pro-gastrin-releasing peptide (ProGRP) and Prolactin (PRL) have high sensitivity from 77.05% to 96.72% and accuracy from 89.71% to 97.79% as well as their cut off which approved the aim of this study. However, other indicators (CEA, SCCA and CYFRA21-1) have low sensitivity from 62.49 % to 87.31% and accuracy between 71.27 % and 88.43 %. Meanwhile, CT scan has high sensitivity around 83% with specificity about 71%. Conclusion Alternative biomarkers (CEA, SCCA, CYFRA21-1, ProGRP, Prolactin and CKAP4) are considered as prospective markers to differentiate healthy individuals from lung cancer disease by their significant results in early stages detection. Thence, if serum CEA, SCCA, CYFRA21-1, ProGRP, Prolactin and CKAP4 are measured together, it would be the precise criterion of early levels of lung cancer rather than the lung biopsy definitely. However, further researches are needed to prove more reliable noninvasive markers of lung cancer.

Establishment of fluorescence immunochromatography detection for cytoskeleton-associated protein 4

A rapid and simple method to detect tumor markers in liver cancer was established by combining immunochromatography technique with fluorescent microsphere labeling. According to the principle of double antibody sandwich, the cytoskeleton-associated protein 4 (CKAP4) paired antibody was used as the labeled and coated antibody, and the goat anti-rabbit polyclonal antibody was used as the quality control line coated antibody in the preparation of the CKAP4 fluorescent immunochromatographic test strips. After the preparation, the test strips were evaluated on various performance indicators, such as linearity, precision and stability. The CKAP4 immunochromatographic strip prepared by time-resolved fluorescent microspheres had high sensitivity, and good specificity. Its precision was within 15%, recovery between 85% and 115%, and linear range between 25 and 1 000 pg/mL. The test strip could be kept stable at 37 °C for 20 days, and it correlated well with commercial ELISA kits. The CKAP4 fluorescence immunochromatography method can quantitatively detect the content of CKAP4 in serum. Furthermore, it is rapid, sensitive, simple, economical and single-person operation. This method has the potential of becoming a new method for the diagnosis and treatment of liver cancer.

CDR1as/miR-7/CKAP4 axis contributes to the pathogenesis of abdominal aortic aneurysm by regulating the proliferation and apoptosis of primary vascular smooth muscle cells.

Abdominal aortic aneurysm (AAA) is characterized as dilation of the aortic wall. Dysregulation of vascular smooth muscle cells (VSMCs) can contribute to the development of this phenotype. Circular RNAs and microRNAs (miRNAs) can regulate the proliferation and apoptosis of VSMCs. This present study aimed to identify the mechanisms of action behind the regulation of cerebellar degeneration-related protein 1 antisense RNA (CDR1as)/miRNA (miR)-7 in VSMCs. The expression levels of miR-7 were upregulated, whereas the levels of CDR1as and cytoskeleton-associated protein 4 (CKAP4) were downregulated in aortic specimens obtained from 10 patients who underwent surgery for AAA compared with aortic specimens from 10 control patients who underwent coronary artery bypass surgery. The molecular mechanism of action of CDR1as/miR-7 was investigated in primary VSMCs. The results of Cell Counting kit-8 and cell growth curve assays revealed that overexpression of CDR1as and knockdown of miR-7, increased VSMC proliferation, whereas knockdown of CDR1as and overexpression of miR-7 suppressed VSMC proliferation. In addition, overexpression of CDR1as and knockdown of miR-7, suppressed apoptosis in VSMCs, indicated by the decreased levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) activity, whereas knockdown of CDR1as and overexpression of miR-7 exhibited the opposite effects. The results of luciferase reporter and biotin pull-down assays confirmed that CDR1as directly bound to miR-7 and suppressed its expression. Additionally, the CDR1as-induced proliferation and suppressed apoptosis was reversed by the overexpression of miR-7. Furthermore, luciferase reporter, reverse transcription-quantitative PCR and western blot assays revealed that miR-7 directly targeted CKAP4 and suppressed its expression. Additionally, the miR-7-suppressed proliferation and increased ROS and LDH activity were reversed by the overexpression of CKAP4. CDR1as also decreased caspase 3/7 activity, which was reversed by miR-7 mimics. miR-7 increased the activity of caspase 3/7, which was again reversed by the overexpression of CKAP4. Therefore, CDR1as, miR-7 and CKAP4 may act in the same pathway to regulate VSMC proliferation and apoptosis.

CKAP4 Regulates Cell Migration via the Interaction with and Recycling of Integrin.

Cytoskeleton-associated protein 4 (CKAP4) is an endoplasmic reticulum protein that is also present in the cell surface membrane, where it acts as a receptor for Dickkopf1 (DKK1). In this study, we found that CKAP4 interacts with β1 integrin and controls the recycling of α5β1 integrin independently of DKK1. In S2-CP8 cells, knockdown of CKAP4 but not DKK1 enlarged the size of cell adhesion sites and enhanced cell adhesion to fibronectin, resulting in decreased cell migration. When CKAP4 was depleted, the levels of α5 but not β1 integrin were increased in the cell surface membrane. A similar phenotype was observed in other cells expressing low levels of DKK1. In S2-CP8 cells, α5 integrin was trafficked with β1 integrin and CKAP4 to the lysosome or recycled with β1 integrin. In CKAP4-depleted cells, the internalization of α5β1 integrin was unchanged, but its recycling was upregulated. Knockdown of sorting nexin 17 (SNX17), a mediator of integrin recycling, abrogated the increased α5 integrin levels caused by CKAP4 knockdown. CKAP4 bound to SNX17, and its knockdown enhanced the recruitment of α5β1 integrin to SNX17. These results suggest that CKAP4 suppresses the recycling of α5β1 integrin and coordinates cell adhesion sites and migration independently of DKK1.

SUN-134 CYTOSKELETON-ASSOCIATED PROTEIN 4 (CKAP4), A NEW PLAYER IN THE REGULATION OF MESANGIAL CELL PROLIFERATION

Cytoskeleton-associated protein 4 (CKAP4) has recently attracted attention within cancer research, where its overexpression has been associated with increased lung, pancreatic and kidney cancer cell proliferation and a worsened prognosis. However, the potential role of CKAP4 in mesangioproliferative diseases such as IgA nephropathy (IgAN) and diabetic nephropathy (DN) has not previously been studied. The main growth factor known to induce proliferation in mesangial cells is PDGF, and the mesangial cells are the only glomerular cells known to express PDGF receptor beta (PDGFRβ). PDGF is normally secreted by the glomerular endothelial cells as well as the mesangial cells themselves resulting in a paracrine and autocrine regulation of mesangial cell proliferation. Increased PDGF levels in the glomeruli leads to mesangial proliferation as seen in DN and IgAN. Our hypothesis is that CKAP4 is involved in the regulation of mesangial proliferation. Expression of CKAP4 in glomeruli was investigated using immunofluorescence and immunoEM on human renal sections. CKAP4 was silenced using shRNA in primary human mesangial cells. Westernblot and masspectrometry was used to study protein expression and BrdU proliferation assay to determine proliferation. In order to understand protein interaction co-immunoprecipitation was used. Finally, two different mouse models of DN was used to investigate CKAP4 expression in disease. Immunohistochemistry as well as immunoEM showed expression of CKAP4 in glomerular mesangial cells and in the cell bodies of podocytes. Silencing CKAP4 resulted in an abated mitogenic response to the growth factor PDGF-BB, and further experiments revealed a significantly decreased expression of the PDGF receptor, both on RNA and protein level. Preliminary results from co-immunoprecipitation experiments indicate that PDGFRβ and CKAP4 interact. Analysis of the total protein content in the silenced cells using mass spectrometry showed only small or no changes in expression of other growth factor receptors such as EGFR and TGFbRs. In two different mouse models of DN CKAP4 was found to be significantly upregulated in later stages of disease with established mesangial expansion and other key features of the disease. We here report for the first time that CKAP4 is expressed in mesangial cells and that the expression of CKAP4 influences the cellular proliferative response to PDGF-BB and the expression of PDGFRβ. CKAP4 has previously been reported to interact with the EGF receptor on the cell surface of hepatocellular carcinomas and thereby influencing the growth of these cells. The EGF receptor and the PDGF receptor has several similarities and are tyrosine kinase receptors. We suggest that the interaction between the PDGF receptor and CKAP4 resembles the one of CKAP4 and EGFR. CKAP4 could be a potential mediator and target of mesangial proliferation in glomerular kidney disease.

Loss of Cytoskeleton‐Associated Protein 4 (CKAP4) Expression Enhances α5β1‐mediated Bladder Cancer Cell Adhesion and Migration

The transmembrane protein cytoskeleton‐associated protein 4 (CKAP4) plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. CKAP4 has also been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. Recent data indicates that loss of CKAP4 expression leads to remodeling of the actin cytoskeleton, including disruption of actin stress fibers, and altered expression of genes known to promote cell motility and dynamic changes in the cytoskeleton. Among these genes, β1‐integrin expression was dramatically increased in response to CKAP4 silencing. Given the importance of tumor cell adhesion and migration for cancer invasion and metastasis, it is important to understand how CKAP4, through modulation of integrin functions, acts to regulate these cellular processes. To this end, we took a loss‐of‐function approach where CKAP4 expression was knocked down by means of siRNA in the human bladder carcinoma cell line T24. Western blot analysis revealed that CKAP4 knockdown induced β1‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Because β1‐integrin associates with α5‐integrin to form the fibronectin receptor, we investigated whether α5‐integrin protein levels are affected by CKAP4 knockdown. Western blot analysis revealed that CKAP4 knockdown induced α5‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Importantly, loss of CKAP4 led to increased surface expression of β1 and α5 integrin and increased cell adhesion and migration on fibronectin as measured by flow cytometry and electric cell‐substrate impedance sensing, respectively. Activation of ER stress and AMPK signaling pathways was also observed in CKAP4‐silenced bladder carcinoma cells. Our data indicate that CKAP4 knockdown mediates adhesion and migration of human bladder cancer cells through transcriptional up‐regulation of α5β1 integrin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CKAP4, a DKK1 Receptor, Is a Biomarker in Exosomes Derived from Pancreatic Cancer and a Molecular Target for Therapy.

The survival rate of pancreatic ductal adenocarcinoma (PDAC) is poor; thus, novel molecularly targeted therapy and companion diagnostics are required. We asked whether cytoskeleton-associated protein 4 (CKAP4), a novel Dickkopf1 (DKK1) receptor, is a candidate for PDAC diagnosis and therapy.Experimental Design: Whether CKAP4 can be secreted with small extracellular vesicles (SEV) from PDAC cells was examined. It was also investigated whether CKAP4 can be detected in sera from patients with PDAC by ELISA using newly generated anti-CKAP4 mAbs and whether anti-CKAP4 mAbs can show antitumor activity in vivo. CKAP4 was secreted with SEVs from PDAC cells, and the SEVs exhibited the characteristics of exosomes. The secretion of CKAP4-containing exosomes was mediated by DKK1-dependent endocytosis routes and required exosome biogenesis molecules. Two ELISAs capable of detecting tumor-secreted CKAP4 were developed. The serum CKAP4 levels were higher in patients with PDAC than healthy control individuals. CKAP4 was highly detected in the sera of pancreatic tumor-bearing xenografted mice and patients with PDAC, whereas CKAP4 was barely detectable in sera from normal mice and postoperative patients. Anti-CKAP4 mAbs with different epitopes demonstrated the inhibitory activities for the binding of DKK1 and CKAP4, AKT activity, and proliferation and migration of PDAC cells. Anti-CKAP4 mAbs also suppressed xenograft tumor formation in immunodeficient mice and extended the survival of mice receiving intraperitoneal or orthotopic injection of PDAC cells. CKAP4 secreted in exosomes may represent a biomarker for PDAC. Anti-CKAP4 mAbs can contribute to the development of novel diagnostic methods and therapeutics.

Serum cytoskeleton-associated protein 4 as a biomarker for the diagnosis of hepatocellular carcinoma.

BackgroundAlpha-fetoprotein (AFP) is the most commonly applied biomarker for diagnosis of hepatocellular carcinoma (HCC), but the low sensitivity and specificity limit its clinical application. Cytoskeleton-associated protein 4 (CKAP4) is a novel oncogenic protein involved in the development and progression of HCC. This study aimed to evaluate whether measurement of circulating CKAP4 could improve diagnostic accuracy for HCC.MethodsWe analyzed data for patients with HCC, chronic hepatitis B infection, and cirrhosis and healthy controls (n=100 in each group), recruited from two centers between July 2013 and December 2015. Circulating levels of CKAP4 were measured with commercial enzyme-linked immunosorbent assay kits. Receiver operating characteristics were used to evaluate diagnostic accuracy.ResultsSerum concentrations of CKAP4 were significantly elevated in the HCC group, in comparison with the three control groups (all P<0.001). The combined biomarker panel (AFP and CKAP4), created by binary logistic regression, presented better performance (area under the curve [AUC] 0.936, 95% CI [0.908–0.965], sensitivity 0.800, specificity 0.963) than AFP (AUC 0.875 [0.835–0.914], sensitivity 0.930, specificity 0.430, P=0.001) or CKAP4 (AUC 0.821 [0.776–0.866], sensitivity 0.790, specificity 0.670, P<0.001) alone to identify HCC, even though CKAP4 alone was not better than AFP (P=0.093). Furthermore, the combined panel also presented a better performance even in identifying early HCC (AUC 0.922 [0.833–0.961]).ConclusionSerum CKAP4 is a novel biomarker for HCC, and it could complement AFP in improving diagnostic accuracy.

P63-Dependent Dickkopf3 Expression Promotes Esophageal Cancer Cell Proliferation via CKAP4.

Dickkopf3 (DKK3) is a secretory protein that belongs to the DKK family, but exhibits structural divergence from other family members, and its corresponding receptors remain to be identified. Although DKK3 has been shown to have oncogenic functions in certain cancer types, the underlying mechanism by which DKK3 promotes tumorigenesis remains to be clarified. We show here that DKK3 stimulates esophageal cancer cell proliferation via cytoskeleton-associated protein 4 (CKAP4), which acts as a receptor for DKK3. DKK3 was expressed in approximately 50% of tumor lesions of esophageal squamous cell carcinoma (ESCC) cases; simultaneous expression of DKK3 and CKAP4 was associated with poor prognosis. Anti-CKAP4 antibody inhibited both binding of DKK3 to CKAP4 and xenograft tumor formation induced by ESCC cells. p63, a p53-related transcriptional factor frequently amplified in ESCC, bound to the upstream region of the DKK3 gene. Knockdown of p63 decreased DKK3 expression in ESCC cells, and reexpression of DKK3 partially rescued cell proliferation in p63-depleted ESCC cells. Expression of ΔNp63α and DKK3 increased the size of tumor-like esophageal organoids, and anti-CKAP4 antibody inhibited growth of esophageal organoids. Taken together, these results suggest that the DKK3-CKAP4 axis might serve as a novel molecular target for ESCC.Significance: In esophageal cancer, findings identify DKK3 as a poor prognostic indicator and demonstrate CKAP4 inhibition as an effective therapeutic strategy. Cancer Res; 78(21); 6107-20. ©2018 AACR.

MicroRNA-671-3p promotes proliferation and migration of glioma cells via targeting CKAP4.

Background and objectiveGlioma is one of the most aggressive and malignant cancers originating from the human brain. Increasing evidence suggests that aberrant expression of microRNAs (miRNAs) frequently occurs in glioma and miRNAs are critical regulators of glioma. miR-671 has recently been revealed to be a novel miRNA that plays a vital role in human glioblastoma multiforme. However, the functional role and underlying mechanisms of miR-671-3p require further analysis.Materials and methodsWestern blot and fluorescence quantitative PCR were used to assess the expression of cytoskeleton-associated protein 4 (CKAP4) and miR-671-3p, respectively. A Cell Counting Kit-8 (CCK-8) assay and a Boyden chamber assay were used to detect the proliferative and migratory abilities of glioma cells. A luciferase assay was used to determine the target gene of miR-671-3p. Apoptosis was analyzed by flow cytometry.ResultsOur results revealed that overexpression of miR-671-3p promoted cell proliferation and migration in vitro. Meanwhile, forced expression of miR-671-3p reduced apoptosis. In contrast, inhibition of miR-671-3p had the opposite effects. We also identified CKAP4 to be a direct target of miR-671-3p. The expression levels of CKAP4 were decreased in clinical samples and inversely correlated with miR-671-3p expression levels. Ectopic expression of CKAP4 reversed the promotive activity of miR-671-3p in the proliferation and migration and enhanced apoptosis.ConclusionOur study demonstrates that miR-671-3p is a predominant positive regulator of glioma progression, thus providing new insights into the molecular mechanisms of glioma development. The findings suggest that the miR-6713p/CKAP4 axis may serve as a potential therapeutic target or biomarker in glioma.

A Succinate Ether Derivative of Tocotrienol Enhances Dickkopf-1 Gene Expression through Epigenetic Alterations in Malignant Mesothelioma Cells

Background: Wnt signaling plays an essential role in tumor cell growth, including the development of malignant mesothelioma (MM). Epigenetic silencing of negative Wnt regulators leading to constitutive Wnt signaling has been observed in various cancers and warrants further attention. We have reported that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic effects in MM cells. Thus, in this study, we investigated whether the anti-MM effect of T3E could be mediated via the epigenetic alteration of the Wnt antagonist gene, Dickkopf-1 (DKK1). Methods: WST-1 and cell analyzers were employed to analyze the effects of T3E on cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR and Western blot were performed to evaluate the expression at mRNA and protein levels. Methylation status and epigenetic modifications of DKK1’s promoter regions after T3E treatment in MM cells were studied using methylation-specific PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown ­(siRNA), and specific inhibitors, were used to validate DKK1 as a target of T3E. Results: T3E markedly impaired MM cell viability, increased the expression of phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, which promotes cell proliferation as a Wnt-independent DKK1 receptor was inhibited by T3E. Silencing CKAP4 by ­siRNA did not appear to directly affect MM cell viability, thereby indicating that expression of both DKK1 and CKAP4 is required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases (DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells leads to increased DKK1 expression, thereby promoting tumor growth inhibition. MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells following T3E treatment is positively correlated with the methylation status of its promoter; T3E decreased DNA methylation and increased histone acetylation. Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation activity, whereas no effects were observed on histone H3K9 and H3K27. Conclusions: Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.

Cytoskeleton-Associated Protein 4 Is a Novel Serodiagnostic Marker for Lung Cancer

Our aim was to develop a serodiagnostic marker for lung cancer. Monoclonal antibodies were generated, and one antibody designated as KU-Lu-1, recognizing cytoskeleton-associated protein 4 (CKAP4), was studied further. To evaluate the utility of KU-Lu-1 antibody as a serodiagnostic marker for lung cancer, reverse-phase protein array analysis was performed with sera of 271 lung cancer patients and 100 healthy controls. CKAP4 was detected in lung cancer cells and tissues, and its secretion into the culture supernatant was also confirmed. The serum CKAP4 levels of lung cancer patients were significantly higher than those of healthy controls (P<0.0001), and the area under the curve of receiver-operating characteristic curve analysis was 0.890, with 81.1% sensitivity and 86.0% specificity. Furthermore, the serum CKAP4 levels were also higher in patients with stage I adenocarcinoma or squamous cell carcinoma than in healthy controls (P<0.0001). Serum CKAP4 levels may differentiate lung cancer patients from healthy controls, and they may be detected early even in stage I non-small cell lung cancer. Serum CKAP4 levels were also significantly higher in lung cancer patients than in healthy controls in the validation set (P<0.0001). The present results provide evidence that CKAP4 may be a novel early serodiagnostic marker for lung cancer.

Activation of the Dickkopf1-CKAP4 pathway is associated with poor prognosis of esophageal cancer and anti-CKAP4 antibody may be a new therapeutic drug.

Aberrant expression of the secretory protein Dickkopf1 (DKK1) is associated with poor prognosis of esophageal squamous cell carcinoma (ESCC), but the underlying mechanism of how DKK1 is involved in aggressiveness of ESCC is not clear. In this study, we show that cytoskeleton-associated protein 4 (CKAP4) functions as a DKK1 receptor in ESCC cells. Immunohistochemical analyses of ESCC revealed that both DKK1 and CKAP4 are minimally expressed in associated normal esophageal squamous mucosa of non-tumor regions, but strongly expressed in tumor lesions. Forty-six of 119 cases (38.7%) were positive for both DKK1 and CKAP4. Those expressing both proteins showed poor prognosis and relapse-free survival. Multivariate analysis demonstrated that expression of both proteins was the poor prognostic factor. The Cancer Genome Atlas data set indicated that the mRNA levels of DKK1 and CKAP4 are significantly elevated in the tumor lesions compared to non-tumor regions. DKK1 bound to CKAP4 at endogenous levels. DKK1 induced the internalization of CKAP4 from and its recycling to the plasma membrane. AKT was activated in ESCC cells in which DKK1 was highly expressed and CKAP4 was localized to the plasma membrane. Knockdown of either DKK1 or CKAP4 inhibited AKT activity and cell proliferation in vitro and xenograft tumor formation. Wild-type CKAP4 or DKK1, but not a DKK1 mutant that was unable to bind to CKAP4, rescued phenotypes induced by CKAP4 or DKK1 knockdown, respectively. The anti-CKAP4 antibody also inhibited AKT activity and suppressed xenograft tumor formation. In contrast, in ESCC cells in which DKK1 was marginally expressed, knockdown of CKAP4 or anti-CKAP4 antibody affected neither AKT activity nor cell proliferation. These findings suggest that the DKK1-CKAP4 pathway promotes ESCC cell proliferation and that CKAP4 might represent a novel therapeutic target for ESCCs expressing both DKK1 and CKAP4.

Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain

BackgroundAntiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4.ResultsWe immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127–602) or the extracellular domain plus the transmembrane domain (Aa 106–602). Positive binding was detected to both CKAP4126–602 and CKAP4106–602, suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4127–360 and CKAP4361–524 exhibited a fast association rate (kon) and a slow dissociation rate (koff), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127–524 region of the CKAP4 extracellular domain.ConclusionsWe determined that the CKAP4127–360 and CKAP4361–524 mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.

Peripheral blood RNA gene expression in children with pneumococcal meningitis: a prospective case–control study

IntroductionInvasive pneumococcal disease (IPD), caused by Streptococcus pneumoniae, is a leading cause of pneumonia, meningitis and septicaemia worldwide, with increased morbidity and mortality in HIV-infected children.ObjectivesWe aimed to compare peripheral...

Cytoskeleton‐Associated Protein 4 (CKAP4) Knockdown Disrupts the Actin Filament Network in Bladder Carcinoma Cells

Cytoskeleton‐associated protein 4 (CKAP4) is a 63kDa, non‐glycosylated type II integral membrane protein that plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. More recently, CKAP4 has been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. While research shows that CKAP4 interacts directly with the microtubule network, its potential role within the actin filament network has not been previously studied. To explore this putative role, we used RNA interference to reduce CKAP4 expression in T24 bladder carcinoma cells and then fixed and stained the actin cytoskeleton using phalloidin‐488. We observed a marked difference in actin filament morphology and a disruption of the actin filament network in cells treated with CKAP4 siRNA versus cells treated with control siRNA or vehicle alone; moreover, CKAP4 knock down generated a significant reduction in actin polymerization. To understand the observed phenotypic changes, we performed differential gene expression analysis using quantitative PCR. Our data showed that CKAP4 knock down led to an increase in the expression of genes known to promote cell motility and dynamic changes in the cytoskeleton with a concomitant reduction in the expression of genes involved in stress fiber formation. Western blot analysis of selected, differentially expressed proteins supported the gene expression data. Collectively, these results suggest that CKAP4 may play an important role in regulating the actin cytoskeleton.