GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

CKAP4 is a potential exosomal biomarker and therapeutic target for lung cancer.

Background: Globally, lung cancer causes the most cancer death. While molecular therapy progress, including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), has provided remarkable therapeutic effects, some patients remain resistant to these therapies and therefore new target development is required. Cytoskeleton-associated membrane protein 4 (CKAP4) is a receptor of the secretory protein Dickkopf-1 (DKK1) and the binding of DKK1 to CKAP4 promotes tumor growth via Ak strain transforming (AKT) activation. We investigated if CKAP4 functions as a diagnostic biomarker and molecular therapeutic target for lung cancer. Methods: CKAP4 secretion with exosomes from lung cancer cells and the effect of CKAP4 palmitoylation on its trafficking to the exosomes were examined. Serum CKAP4 levels were measured in mouse xenograft models, and 92 lung cancer patients and age- and sex-matched healthy controls (HCs). The lung cancer tissues were immunohistochemically stained for DKK1 and CKAP4, and their correlation with prognosis and serum CKAP4 levels were investigated. Roles of CKAP4 in the lung cancer cell proliferation were examined, and the effects of the combination of an anti-CKAP4 antibody and osimertinib, a third generation TKI, on anti-tumor activity were tested using in vitro and in vivo experiments. Results: CKAP4 was released from lung cancer cells with exosomes, and its trafficking to exosomes was regulated by palmitoylation. CKAP4 was detected in sera from mice inoculated with lung cancer cells overexpressing CKAP4. In 92 lung cancer patients, positive DKK1 and CKAP4 expression patients showed worse prognoses. Serum CKAP4 positivity was higher in lung cancer patients than in HCs. After surgical operation, serum CKAP4 levels were decreased. CKAP4 overexpression in lung cancer cells promoted in vitro cell proliferation and in vivo subcutaneous tumor growth, which were inhibited by an anti-CKAP4 antibody. Moreover, treatment with this antibody or osimertinib, a third generation TKI, inhibited AKT activity, sphere formation, and xenograft tumor growth in lung cancer cells harboring EGFR mutations and expressing both DKK1 and CKAP4, while their combination showed stronger inhibition. Conclusions: CKAP4 may represent a novel biomarker and molecular target for lung cancer, and combination therapy with an anti-CKAP4 antibody and osimertinib could provide a new lung cancer therapeutic strategy.

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.

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.

The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers.

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5'-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.

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.

Dynamic palmitoylation controls the microdomain localization of the DKK1 receptors CKAP4 and LRP6.

Dickkopf1 (DKK1) was originally identified as an antagonist of Wnt signaling that binds to and induces the clathrin-mediated endocytosis of the Wnt coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). DKK1 also binds to cytoskeleton-associated protein 4 (CKAP4), which was originally identified as an endoplasmic reticulum (ER) protein but also functions at the plasma membrane as a receptor for various ligands. The DKK1-CKAP4 pathway is activated in several human cancers and promotes cell proliferation by activating signaling through the kinases PI3K and AKT. We found that both CKAP4 and LRP6 primarily localized to detergent-resistant membrane (DRM) fractions of the plasma membrane in a palmitoylation-dependent manner and that palmitoylation of CKAP4 was required for it to promote cell proliferation. DKK1 induced the depalmitoylation of both CKAP4 and LRP6 by acylprotein thioesterases (APTs), resulting in their translocation to the non-DRM fractions. Moreover, DKK1-dependent depalmitoylation of both receptors required activation of the PI3K-AKT pathway. DKK1 simultaneously bound CKAP4 and LRP6, resulting in the formation of a ternary complex. LRP5/6 knockdown decreased DKK1-dependent AKT activation and cancer cell proliferation through CKAP4, whereas CKAP4 knockdown did not affect DKK1-dependent inhibition of Wnt signaling through LRP5/6. These results indicate that the palmitoylation states of CKAP4 and LRP6 play important roles in their signaling and that LRP5/6 enhance DKK1-CKAP4 signaling.

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

PURPOSE: 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. RESULTS: 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. CONCLUSIONS: CKAP4 secreted in exosomes may represent a biomarker for PDAC. Anti-CKAP4 mAbs can contribute to the development of novel diagnostic methods and therapeutics.

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.

Cold snare polypectomy reduced delayed postpolypectomy bleeding compared with conventional hot polypectomy: a propensity score-matching analysis.

BACKGROUND AND STUDY AIMS: Cold snare polypectomy (CSP) for small colorectal polyps has lower incidence of adverse events, especially delayed postpolypectomy bleeding (DPPB). However, few data are available on comparisons of the incidence of DPPB of CSP and hot polypectomy (HP). The aim of this study was to evaluate the incidence of DPPB after CSP and compare it with that of HP. A propensity score model was used as a secondary analysis. PATIENTS AND METHODS: This was a retrospective cohort study conducted in a single municipal hospital. We identified 539 patients with colorectal polyps from 2 mm to 11 mm in size who underwent CSP (804 polyps in 330 patients) or HP (530 polyps in 209 patients) between July 2013 and June 2015. RESULTS: There were no cases of DPPB in the CSP group. Conversely, DPPB occurred in 4 patients (1.9 %) after HP, resulting in a significant difference between the CSP and HP groups (0.008 % vs 0 %, P  = 0.02). Propensity score-matching analysis created 402 matched pairs, yielding a significantly higher DPPB rate in the HP group than CSP group (0.02 % vs 0 %, P  = 0.04). However, significantly more patients in the CSP group had unclear horizontal margins that precluded assessment (83 vs 38 cases, P  < 0.001). The retrieval failure rate was significantly higher in the CSP group than in the HP group (3 % vs 0.7 %, P  = 0.01). CONCLUSIONS: DPPB was less frequent with CSP than HP, as selected by the propensity score-matching model. Our findings indicate that CSP is recommended polypectomy in daily clinical setting. However, special care should be taken during polyp retrieval and horizontal margin assessment, and these issues could be taken into account in follow-up after CSP.

Gastric atrophy and xanthelasma are markers for predicting the development of early gastric cancer.

BACKGROUND: The significance of gastric xanthelasma in relation to gastric cancer still remains unclear. We investigated whether gastric xanthelasma would be a useful marker for predicting the development of early gastric cancer. METHODS: A total of 1823 patients who underwent a medical health checkup were enrolled. We examined the relationship between gastric xanthelasma and various clinical features, and in an endoscopic follow-up study investigated whether the presence of gastric xanthelasma was predictive of the development of early gastric cancer. RESULTS: In the initial endoscopic examination, gastric xanthelasma was detected in 107 (5.9 %) of the 1823 patients. The presence of gastric xanthelasma was significantly associated with age ≥65 years, male gender, open-type atrophy, and the presence of diabetes mellitus (DM) (p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). During the endoscopic follow-up period, early gastric cancer was found in 29 (1.6 %) of the 1823 patients. Gastric cancer occurred in 15 (14.0 %) of 107 patients with gastric xanthelasma, whereas it occurred in 14 (0.8 %) of 1716 patients without (p < 0.0001). Multivariate analysis revealed that open-type atrophy and gastric xanthelasma were independently related to the development of gastric cancer (odds ratio 7.19 [2.50-20.83]; p = 0.0003 and 5.85 [2.67-12.82]; p < 0.0001, respectively). The presence of gastric xanthelasma was significantly predictive of gastric cancer development even in the selected high-risk groups with open-type atrophy or DM (p < 0.0001 or p < 0.0001, respectively). CONCLUSIONS: Gastric xanthelasma is a useful marker for predicting the development of gastric cancer.