DKK1 Activates the PI3K/AKT Pathway via CKAP4 to Balance the Inhibitory Effect on Wnt/ß-Catenin Signaling and Regulates Wnt3a-Induced MSC Migration.

Wnt/ß-catenin signaling plays a crucial role in the migration of mesenchymal stem cells (MSCs). However, our study has revealed an intriguing phenomenon where Dickkopf-1 (DKK1), an inhibitor of Wnt/ß-catenin signaling, promotes MSC migration at certain concentrations ranging from 25 to 100 ng/mL while inhibiting Wnt3a-induced MSC migration at a higher concentration (400 ng/mL). Interestingly, DKK1 consistently inhibited Wnt3a-induced phosphorylation of LRP6 at all concentrations. We further identified cytoskeleton-associated protein 4 (CKAP4), another DKK1 receptor, to be localized on the cell membrane of MSCs. Overexpressing the CRD2 deletion mutant of DKK1 (ΔCRD2), which selectively binds to CKAP4, promoted the accumulation of active ß-catenin (ABC), the phosphorylation of AKT (Ser473) and the migration of MSCs, suggesting that DKK1 may activate Wnt/ß-catenin signaling via the CKAP4/PI3K/AKT cascade. We also investigated the effect of the CKAP4 intracellular domain mutant (CKAP4-P/A) that failed to activate the PI3K/AKT pathway and found that CKAP4-P/A suppressed DKK1 (100 ng/mL)-induced AKT activation, ABC accumulation, and MSC migration. Moreover, CKAP4-P/A significantly weakened the inhibitory effects of DKK1 (400 ng/mL) on Wnt3a-induced MSC migration and Wnt/ß-catenin signaling. Based on these findings, we propose that DKK1 may activate the PI3K/AKT pathway via CKAP4 to balance the inhibitory effect on Wnt/ß-catenin signaling and thus regulate Wnt3a-induced migration of MSCs. Our study reveals a previously unrecognized role of DKK1 in regulating MSC migration, highlighting the importance of CKAP4 and PI3K/AKT pathways in this process.

Elucidation of CKAP4-remodeled cell mechanics in driving metastasis of bladder cancer through aptamer-based target discovery.

Metastasis contributes to the dismal prognosis of bladder cancer (BLCA). The mechanical status of the cell membrane is expected to mirror the ability of cell migration to promote cancer metastasis. However, the mechanical characteristics and underlying molecular profile associated with BLCA metastasis remain obscure. To study the unique cellular architecture and traits associated with cell migration, using a process called cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) we generated an aptamer-based molecular probe, termed spl3c, which identified cytoskeleton-associated protein 4 (CKAP4). CKAP4 was associated with tumor metastasis in BLCA, but we also found it to be a mechanical regulator of BLCA cells through the maintenance of a central-to-peripheral gradient of stiffness on the cell membrane. Notably, such mechanical traits were transportable through exosome-mediated intercellular CKAP4 trafficking, leading to significant enhancement of migration in recipient cells and, consequently, aggravating metastatic potential in vivo. Taken together, our study shows the robustness of this aptamer-based molecular tool for biomarker discovery, revealing the dominance of a CKAP4-induced central-to-peripheral gradient of membrane stiffness that benefits cell migration and delineating the role of exosomes in mediating mechanical signaling in BLCA metastasis.

Dickkopf signaling, beyond Wnt-mediated biology.

Dickkopf1 (DKK1) was originally identified as a secreted protein that antagonizes Wnt signaling. Although DKK1 is essential for the developmental process, its functions in postnatal and adult life are unclear. However, evidence is accumulating that DKK1 is involved in tumorigenesis in a manner unrelated to Wnt signaling. In addition, recent studies have revealed that DKK1 may control immune reactions, although the relationship of this to Wnt signaling is unknown. Other DKK family members, DKK2-4, are likely to have their own functions. Here, we review the possible novel functions of DKKs. We summarize the characteristics of receptors of DKKs and the signaling mechanisms through DKKs and their receptors, provide evidence showing that DKKs are involved in tumor aggressiveness independently of Wnt signaling, and emphasize promising cancer therapies targeting DKKs and receptors. Lastly, we discuss various physiological and pathological processes controlled by DKKs.

Newly developed humanized anti-CKAP4 antibody suppresses pancreatic cancer growth by inhibiting DKK1-CKAP4 signaling.

Cytoskeleton-associated protein 4 (CKAP4) is a cell surface receptor for Dickkopf 1 (DKK1), a secreted protein. The DKK1-CKAP4 pathway is activated in various malignant tumors, including pancreatic, lung, esophageal, and liver cancers, to promote tumor growth. Thus, CKAP4 has been expected to represent a novel molecular target of cancer therapy. Recombinant mouse anti-CKAP4 antibodies were generated based on an original mouse antibody (3F11-2B10) and inhibited DKK1-CKAP4 signaling and xenograft tumor formation induced by pancreatic cancer cells, which was comparable with 3F11-2B10. From the 3F11-2B10 nucleotide sequence, humanized anti-CKAP4 antibody (Hv1Lt1) was subsequently developed. The binding affinity of Hv1Lt1 for CKAP4 was superior to that of 3F11-2B10. Hv1Lt1 inhibited DKK1 binding to CKAP4, AKT activity, and sphere formation of pancreatic cancer cells, which was comparable with 3F11-2B10. Hv1Lt1 also suppressed xenograft tumor formation induced by human pancreatic cancer cells and tumor growth in murine cancer models, in which murine pancreatic cancer organoids were orthotopically transplanted into the pancreas. In resected tumor samples from mice treated with Hv1Lt1, anti-tumor immune reactions were modulated and cytotoxic T cells were highly infiltrated in the tumor microenvironment. Additionally, combination of Hv1Lt1 and other chemotherapy drugs exhibited stronger effects compared with monotherapy. These results suggest that Hv1Lt1 represents a promising anti-cancer therapy.

The Role of Serum Dickkopf1 and CKAP4 Levels in Diagnosing Colorectal Cancer and Measuring the Disease Severity: A Prospective Study.

Background and Objective: Colorectal cancer (CRC) is among the most common types of cancer. Although the disease is treatable in its early stages, five-year survival falls below 20% in the later stages. CEA and CA19-9 are tumor markers used in the diagnosis and follow-up of the disease in clinical practice; however, their diagnostic effectiveness is insufficient. Therefore, the identification of biomarkers that can be easily studied from serum and can diagnose CRC and determine its severity is highly important. In this context, dickkopf1 (DKK1) and cytoskeleton-associated protein 4 (CKAP4) are both promising biomarkers. Materials and Methods: Serum DKK1 and CKAP4 levels were measured in 55 patients with CRC and 40 healthy controls. The patients with CRC were divided into groups based on pathological stages and histological differentiation. The serum levels of both proteins in patients with CRC were measured preoperatively and 10 and 30 days postoperatively. Results: Serum DKK1 and CKAP4 were significantly higher in the CRC group than in the healthy controls (p < 0.05). Serum levels of both proteins rose in line with the disease stage and grade but decreased following surgical resection. A positive correlation was observed between tumor diameter and protein blood levels. The diagnostic efficacy of DKK1 and CKAP4 in CRC (approximately 95%) was higher than that of markers such as CEA and CA19-9. Conclusions: The DKK1 and CKAP4 serum values of patients with CRC are promising biomarkers. They can potentially be used in CRC management, namely, in the diagnosis and treatment of tumor response access and in tumor aggressiveness prediction.

Formin-like 2 promotes angiogenesis and metastasis of colorectal cancer by regulating the EGFL6/CKAP4/ERK axis.

Increasing evidence indicates that angiogenesis plays a pivotal role in tumor progression. Formin-like 2 (FMNL2) is well-known for promoting metastasis; however, the molecular mechanisms by which FMNL2 promotes angiogenesis in colorectal cancer (CRC) remain unclear. Here, we found that FMNL2 promotes angiogenesis and metastasis of CRC in vitro and in vivo. The GDB/FH3 domain of FMNL2 directly interacts with epidermal growth factor-like protein 6 (EGFL6). Formin-like 2 promotes EGFL6 paracrine signaling by exosomes to regulate angiogenesis in CRC. Cytoskeleton associated protein 4 (CKAP4) is a downstream target of EGFL6 and is involved in CRC angiogenesis. Epidermal growth factor-like protein 6 binds to the N-terminus of CKAP4 to promote the migration of HUVECs by activating the ERK/MMP pathway. These findings suggest that FMNL2 promotes the migration of HUVECs and enhances angiogenesis and tumorigenesis in CRC by regulating the EGFL6/CKAP4/ERK axis. Therefore, the EGFL6/CKAP4/ERK axis could be a candidate therapeutic target for CRC treatment.

DKK1-CKAP4 signal axis promotes hepatocellular carcinoma aggressiveness.

Hepatocellular carcinoma (HCC) is the most prevalent malignant liver neoplasm. Despite the advances in diagnosis and treatment, the prognosis of HCC patients remains poor. Cytoskeleton-associated membrane protein 4 (CKAP4) is a receptor of the glycosylated secretory protein Dickkopf-1 (DKK1), and the DKK1-CKAP4 axis is activated in pancreatic, lung, and esophageal cancer cells. Expression of DKK1 and CKAP4 has been examined in HCC in independent studies that yielded contradictory results. In this study, the relationship between the DKK1-CKAP4 axis and HCC was comprehensively examined. In 412 HCC cases, patients whose tumors were positive for both DKK1 and CKAP4 had a poor prognosis compared to those who were positive for only one of these markers or negative for both. Deletion of either DKK1 or CKAP4 inhibited HCC cell growth. In contrast to WT DKK1, DKK1 lacking the CKAP4 binding region did not rescue the phenotypes caused by DKK1 depletion, suggesting that binding of DKK1 to CKAP4 is required for HCC cell proliferation. Anti-CKAP4 Ab inhibited HCC growth, and its antitumor effect was clearly enhanced when combined with lenvatinib, a multikinase inhibitor. These results indicate that simultaneous expression of DKK1 and CKAP4 is involved in the aggressiveness of HCC, and that the combination of anti-CKAP4 Ab and other therapeutics including lenvatinib could represent a promising strategy for treating advanced HCC.

Circular RNA suppression of vascular smooth muscle apoptosis through the miR-545-3p/CKAP4 axis during abdominal aortic aneurysm formation.

BACKGROUND: Abdominal aortic aneurysms (AAA) are an important cause of cardiovascular deaths. The loss of vascular smooth muscle cells (VSMCs) has been reported to be related to the pathology of AAA. This study focused on investigating the function of circ_0002168 in VSMC apoptosis. METHODS: Levels of genes and proteins were measured by quantitative real-time-polymerase chain reaction (qRT-PCR) and Western blot. The growth of VSMCs was determined by using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry and the evaluation of caspase-3 activity analysis, reactive oxygen species (ROS) production as well as lactate dehydrogenase (LDH) activity. The binding between miR-545-3p and circ_0002168 or Cytoskeleton-associated protein 4 (CKAP4) was confirmed by bioinformatics analysis, dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. RESULTS: Circ_0002168 decreased in the aortic tissues of patients with AAA. Functionally, ectopic overexpression of circ_0002168 dramatically induced proliferation and suppressed apoptosis in VSMCs. Mechanistically, circ_0002168 sequestered miR-545-3p to release CKAP4 expression via the ceRNA mechanism, indicating the circ_0002168/miR-545-3p/CKAP4 feedback loop in VSMCs. Increased miR-545-3p and a decreased CKAP4 expression were observed in patients with AAA. Rescue experiments showed that miR-545-3p reversed the protective effects of circ_0002168 on VSMC proliferation. Moreover, inhibition of miR-545-3p could restrain the apoptosis of VSMCs, which was abolished by CKAP4 silencing. CONCLUSION: Circ_0002168 has a protective effect on VSMC proliferation by regulating the miR-545-3p/CKAP4 axis, adding further understanding of the pathogenesis of AAA and a potential therapeutic approach in AAA management.

DKK3/CKAP4 axis is associated with advanced stage and poorer prognosis in oral cancer.

OBJECTIVE: We previously reported that dickkopf WNT signaling inhibitor 3 (DKK3) would modulate malignant potential of oral squamous cell carcinoma (OSCC) via activating Akt. Recently, cytoskeleton associated protein 4 (CKAP4) functions as receptor of DKK3, which activates Akt in esophageal squamous cell carcinoma, but its expression and function in OSCC were unclear. METHODS: We studied DKK3 and CKAP4 protein expression in OSCC tissue and investigated the correlation between protein expression and clinical data. We also investigated whether antibodies (Ab) for DKK3 or CKAP4 could suppress malignant potential of the cancer cells. RESULTS: DKK3/CKAP4 protein expression was observed in majority of OSCC cases and was associated with significantly higher T-stage and TNM stage. Multivariate analysis revealed that DKK3 and CKAP4 were independent prognostic biomarkers for overall survival (OS) and disease-free survival (DFS), respectively. Survival analyses revealed that DKK3-positive cases and CKAP4-positive cases showed significantly shorter OS and DFS, respectively, and that DKK3/CKAP4 double-negative cases showed significantly favorable prognosis. Both anti-DKK3Ab and anti-CKAP4Ab could suppress cancer cell proliferation, migration, and invasion. CONCLUSION: DKK3/CKAP4 axis is thought to be important in OSCC, and it would be a promising therapeutic target.

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.

Cytoskeleton-associated protein 4 (CKAP4) promotes malignant progression of human gliomas through inhibition of the Hippo signaling pathway.

PURPOSE: Gliomas are the most common and aggressive malignant brain tumors and are associated with high mortality and incidence in humans. Despite rigorous multi-modal therapy, including surgery, chemotherapy and radiotherapy, patients with malignant glioma survive an average of 12-15 months following primary diagnosis. Therefore, new molecular biomarkers are urgently needed for diagnosis and targeted therapy. Here, we find that suppression of CKAP4 might inhibit glioma growth through regulation of Hippo signaling. METHODS: We examined the expression levels of CKAP4 through analysis of RNA sequencing data from GEPIA and CGGA databases. Then, Lentivirus was used to construct stable cell lines with knockout or overexpression of CKAP4. Next, the function of CKAP4 on glioma was investigated in vitro and in an orthotopic brain tumor model in mice. Lastly, luciferase reporter assay, immunofluorescence and immunoblotting were performed to explore the potential mechanism of how CKAP4 affects gliomas. RESULTS: CKAP4 is highly upregulated in glioma and high CKAP4 expressing tumors were associated with poor patient survival. And CKAP4 promotes malignant progression of gliomas via inhibiting Hippo signaling. CONCLUSION: CKAP4 has potential as a promising biomarker and can predict the prognosis of patients with gliomas. And targeting CKAP4 expression may be an effective therapeutic strategy for the treatment of human gliomas.

Altered expression of p63 isoforms and expansion of p63- and club cell secretory protein-positive epithelial cells in the lung as novel features of aging.

Aging is a key contributor for subclinical progression of late-onset lung diseases. Basal, club, and type II alveolar epithelial cells (AECs) are lung epithelial progenitors whose capacities of differentiation are extensively studied. The timely transition of these cells in response to environmental changes helps maintain the intricate organization of lung structure. However, it remains unclear how aging affects their behavior. This paper demonstrates that the protein expression profiles of a type II AEC marker, prosurfactant protein C (pro-SPC), and a basal cell marker, p63, are altered in the lungs of 14-mo-old versus 7- to 9-wk-old mice. Expression of NH2-terminal-truncated forms of p63 (ΔNp63), a basal cell marker, and claudin-10, a club cell marker, in cytoplasmic extracts of lungs of 14-mo-old mice was upregulated. In contrast, nuclear expression of full-length forms of p63 (TAp63) decreases with age. These alterations in protein expression profiles coincide with dramatic changes in lung functions including compliance. Whole tissue lysates of middle-aged versus aged rhesus monkey lungs display similar age-associated alterations in pro-SPC expression. An age-associated decrease of TAp63 in nuclear lysates was observed in aged monkey group. Moreover, the lungs of 14-mo-old versus 7- to 9-wk-old mice display a wider spreading of ΔNp63-positive CCSP-positive bronchiolar epithelial cells. This expansion did not involve upregulation of Ki67, a representative proliferation marker. Collectively, it is postulated that 1) this expansion is secondary to a transition of progenitor cells committed to club cells from ΔNp63-negative to ΔNp63-positive status, and 2) high levels of cytoplasmic ΔNp63 expression trigger club cell migration.

Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation.

BACKGROUND: Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. METHODS: Here, we present a method to obtain high-quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. RESULTS: After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression, we could identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton-associated protein 4 as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Cytoskeleton-associated protein 4 inhibition in activated fibroblasts treated with transforming growth factor ß triggered a greater increase in the expression of genes related to activated fibroblasts compared with control, suggesting a role of cytoskeleton-associated protein 4 in modulating fibroblast activation in the injured heart. CONCLUSIONS: Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.

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

BACKGROUND: Antiproliferative 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. RESULTS: We 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 (k on ) and a slow dissociation rate (k off ), 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. CONCLUSIONS: We 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.

LRRC59 promotes the progression of oral squamous cell carcinoma by interacting with SRP pathway components and enhancing the secretion of CKAP4-containing exosomes.

Background: As a ribosome receptor, LRRC59 was thought to regulate mRNA translation on the ER membrane. Evidence suggests that LRRC59 is overexpressed in a number of human malignancies and is associated with poor prognoses, but its primary biological function in the development of oral squamous cell carcinoma (OSCC) remains obscure. Objective: The purpose of this study is to investigate at the expression changes and functional role of LRRC59 in OSCC. Methods: LRRC59 gene expression and correlation with prognosis of OSCC patients were first examined using the data from The Cancer Genome Atlas (TCGA) databases. Following that, a series of functional experiments, including cell counting kit-8, cell cycle analysis, wound healing assays, and transwell assays, were carried out to analyze the biological roles of LRRC59 in tumor cells. Mechanistically, we employed Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach to isolate and identify protein complexes of LRRC59. Downstream regulatory proteins of LRRC59 were verified through immunoprecipitation and immunofluorescence experiments. Furthermore, we isolated exosomes from OSCC cell supernatant and conducted co-culture experiments to examine the effect of LRRC59 knockdown on OSCC cells. Results: In samples from OSCC patients, LRRC59 was highly expressed and correlated with poor prognoses. Moreover, the gene sets analysis based on TCGA RNA-seq data indicated that LRRC59 seemed to be strongly related with protein secretory and OSCC migration. Upregulated levels of LRRC59 are more prone to lymph node metastasis in OSCC patients. LRRC59 knockdown impaired the ability of OSCC cell proliferation, migration, and invasion invitro. Mechanistically, our TAP-MS data situate LRRC59 in a functional nexus for mRNA translation regulation via interactions with SRP pathway components, translational initiation factors, CRD-mediated mRNA stabilization factors. More importantly, we found that LRRC59 interacted with cytoskeleton-associated protein 4 (CKAP4) and promoted the formation of CKAP4-containing exosomes. We also revealed that the LRRC59-CKAP4 axis was a crucial regulator of CKAP4-containing exosome secretion in OSCC cells for migration and invasion. Conclusions: Therefore, based on our findings, LRRC59 may serve as a potential biomarker for OSCC patients, and LRRC59-induced exosome secretion via the CKAP4 axis may serve as a potential therapeutic target for OSCC.

Promoter hypermethylation-induced downregulation of ITGA7 promotes colorectal cancer proliferation and migration by activating the PI3K/AKT/NF-κB pathway.

We previously reported that integrin alpha 7 (ITGA7) was downregulated in colorectal cancer (CRC) tissues and CRC cell lines and that the lower expression of ITGA7 in CRC tissues was correlated with distant metastasis, suggesting that ITGA7 may function as a suppressor in CRC. The present research was conducted to further investigate the role and mechanisms of ITGA7 in CRC progression. First, bisulfite modification and genomic sequencing (BSP) results showed that the methylation rate of ITGA7 promoter was higher in 10 CRC tissues than in the matched normal tissues. Additionally, 5-Aza-CdR treatment increased ITGA7 expression in CRC cells. Gain-of-function assays revealed the inhibitory role of ITGA7 in CRC cell proliferation and migration. Mechanistically, RNA sequencing, RT-qPCR, and cytoplasm and nuclear separation and rescue assays indicated that knockdown of ITGA7 activated the transcription of MMP9, SETD7, and ADAM15 by enhancing the nuclear translocation of NF-κB. Moreover, CoIP and Western blot suggested a mechanistic model in which ITGA7 binds to CKAP4 to block the interaction of CKAP4 and PI3K p85α and thereby suppress the PI3K/AKT/NF-κB pathway. Accordingly, the current study suggests that ITGA7 functions as a suppressor in CRC progression and that its expression is controlled by promoter methylation.

Disposable Zirconium trisulfide-Reduced graphene oxide modified conducting thread based electrochemical biosensor for lung cancer diagnosis.

Flexible technology in sensors have received much attention in monitoring of human health through various physiological indicators. Thus, it drawn a lot of interest in the development of flexible substrate for the diagnosis of various diseases via analysis of analytes. Present work focusses on the development of ecofriendly, portable, flexible, conducting thread (Th) and used as smart substrate for fabrication of biosensor towards ultrasensitive detection of the lung cancer biomarker (cytoskeleton-associated protein 4; CKAP4). The zirconium trisulfide-reduced graphene oxide nanocomposite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) modified cotton thread based biosensor was fabricated via dip coating method. Next, successive immobilization of monoclonal antibodies of CKAP4 (anti-CKAP4) and bovine serum albumin (BSA) was performed via drop cast approach using fabricated electrode [nZrS3@rGO/PEDOT:PSS/Th]. The response of fabricated electrode (BSA/anti-CKAP4/ZrS3@rGO/PEDOT:PSS/Th) was recorded electrochemically versus CKAP4 concentration via chronoamperometry (CA). The results showed wider linear detection range of 6.25-800 pg mL-1, excellent sensitivity of 85.2 µA[log(pg mL-1)]-1cm-2 with good stability up to 42 days. The response of fabricated biosensor was supported by investigating response of CKAP4 biomarker present in patients of lung cancer (concentration as determined through enzyme-linked immunosorbent assay) and obtained results exhibited excellent correlation with that of standard samples.

Invigorating human MSCs for transplantation therapy via Nrf2/DKK1 co-stimulation in an acute-on-chronic liver failure mouse model.

Background: Since boosting stem cell resilience in stressful environments is critical for the therapeutic efficacy of stem cell-based transplantations in liver disease, this study aimed to establish the efficacy of a transient plasmid-based preconditioning strategy for boosting the capability of mesenchymal stromal cells (MSCs) for anti-inflammation/antioxidant defenses and paracrine actions in recipient hepatocytes. Methods: Human adipose mesenchymal stem cells (hADMSCs) were subjected to transfer, either with or without the nuclear factor erythroid 2-related factor 2 (Nrf2)/Dickkopf1 (DKK1) genes, followed by exposure to TNF-α/H2O2. Mouse models were subjected to acute chronic liver failure (ACLF) and subsequently injected with either transfected or untransfected MSCs. These hADMSCs and ACLF mouse models were used to investigate the interaction between Nrf2/DKK1 and the hepatocyte receptor cytoskeleton-associated protein 4 (CKAP4). Results: Activation of Nrf2 and DKK1 enhanced the anti-stress capacity of MSCs in vitro. In a murine model of ACLF, transient co-overexpression of Nrf2 and DKK1 via plasmid transfection improved MSC resilience against inflammatory and oxidative assaults, boosted MSC transplantation efficacy, and promoted recipient liver regeneration due to a shift from the activation of the anti-regenerative IFN-γ/STAT1 pathway to the pro-regenerative IL-6/STAT3 pathway in the liver. Importantly, the therapeutic benefits of MSC transplantation were nullified when the receptor CKAP4, which interacts with DKK1, was specifically removed from recipient hepatocytes. However, the removal of the another receptor low-density lipoprotein receptor-related protein 6 (LRP6) had no impact on the effectiveness of MSC transplantation. Moreover, in long-term observations, no tumorigenicity was detected in mice following transplantation of transiently preconditioned MSCs. Conclusions: Co-stimulation with Nrf2/DKK1 safely improved the efficacy of human MSC-based therapies in murine models of ACLF through CKAP4-dependent paracrine mechanisms.

Expression of cytoskeleton-associated protein 4 is associated with poor prognosis and metastasis in nasopharyngeal carcinoma.

Cytoskeleton-associated protein 4 (CKAP4) acts as a key transmembrane protein that connects the endoplasmic reticulum (ER) to microtubule dynamics. Researchers have not examined the roles of CKAP4 in nasopharyngeal carcinoma (NPC). The study aimed at evaluating the prognostic value and metastasis-regulating effect of CKAP4 in NPC. CKAP4 protein could be observed in 86.36% of 557 NPC specimens but not in normal nasopharyngeal epithelial tissue. According to immunoblot assays, NPC cell lines presented high CKAP4 expression relative to NP69 immortalized nasopharyngeal epithelial cells. Moreover, CKAP4 was highly expressed at the NPC tumor front and in matched liver, lung, and lymph node metastasis samples. Furthermore, high CKAP4 expression reported poor overall survival (OS) and presented a positive relevance to tumor (T) classification, recurrence, and metastasis. According to multivariate analysis, CKAP4 could independently and negatively predict patients' prognosis. Stable knockdown of CKAP4 expression in NPC cells inhibited cell migration, invasion and metastasis in vitro and in vivo. Moreover, CKAP4 promoted epithelial-mesenchymal transition (EMT) in NPC cells. CKAP4 knockdown was followed by the downregulation of the interstitial marker vimentin, and upregulation of the epithelial marker E-cadherin. In NPC tissues, high CKAP4 expression exhibited a positive relevance to vimentin expression and a negative relevance to E-cadherin expression. In conclusion, CKAP4 is an independent predictor of NPC, and CKAP4 might contribute NPC progression and metastasis, which may be involved in EMT with vimentin and E-cadherin.

CKAP4-mediated activation of FOXM1 via phosphorylation pathways regulates malignant behavior of glioblastoma cells.

OBJECTIVE: CKAP4 (Cytoskeleton Associated Protein 4) has been reported as an important regulator of carcinogenesis. A great deal of uncertainty still surrounds the possible molecular mechanism of CKAP4 involvement in GBM. We aimed to specifically elucidate the putative role of CKAP4 in the development of GBM. METHODS: We identified divergent proteomics landscapes of GBM and adjacent normal tissues using mass spectrometry-based label-free quantification. Bioinformatics analysis of differentially expressed proteins (DEPs) led to the identification of CKAP4 as a hub gene. Based on the Chinese Glioma Genome Atlas data, we characterized the elevated expression of CKAP4 in GBM and developed a prognostic model. The influence of CKAP4 on malignant behavior of GBM was detected in vitro and vivo, as well as its downstream target and signaling pathways. RESULTS: The prognosis model displayed accuracy and reliability for the probability of survival of patients with gliomas. CKAP4 knockdown remarkably reduced the malignant potential of GBM cells, whereas its overexpression reversed these effects in GBM cells and xenograft mice. Moreover, we demonstrated that overexpression of CKAP4 leads to increased FOXM1 (Forkhead Box M1) expression in conjunction with an increased level of AKT and ERK phosphorylation. Inhibition of both pathways had synergistic effects, resulting in greater effectiveness of inhibition. CKAP4 could reverse the deregulation of FOXM1 triggered by inhibition of AKT and ERK signaling. CONCLUSIONS: This is the first study to reveal a CKAP4-FOXM1 signaling cascade that contributes to the malignant phenotype of GBMs. The CKAP4-based prognostic model would facilitate individualized treatment decisions for glioma patients.