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March 27, 2023
Development of biomarker and drug development using CKAP4 as a target in lung cancer
(Prof.A.Kikuchi,et al. in Transl. Lung Cancer Res. )
Overview of the Research:
Professor Akira Kikuchi (Center for Infectious Diseases Education and Research, Osaka University), along with Assistant Professor Ryota Sada and post-graduate student Akihiro Nagoya (Graduate School of Medicine, Osaka University), revealed that CKAP4 becomes a novel target for diagnosis and treatment in lung adenocarcinoma (lung cancer) where the DKK1-CKAP4 signaling pathway is activated. In Japan, the number of deaths due to lung cancer has been increasing every year and exceeded 75,000 in 2020, ranking first in the number of deaths and death rate among all cancers. In worldwide, lung cancer causes 1.8 million deaths, which also ranks first among all cancers. Although immune checkpoint inhibitors and drugs targeting driver mutations including EGF receptor inhibitors and Ras inhibitors have been developed, some cases are unresponsive to them or relapse after treatment, and the development of new molecular targeted therapies is required. In this study, Professor Kikuchi’s group found that CKAP4 becomes a biomarker for the activation of the DKK1-CKAP4 pathway in lung cancer. They also found that the anti-CKAP4 antibody enhances the anti-tumor effect when used in combination with osimertinib, the first-line EGF receptor-mutated lung cancer treatment drug, suggesting the potential for the development of new therapies. These research achievements were published in Translational Lung Cancer Research.
Fig.
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Credit: Credit: Kikuchi et al.
Research Background
Dickkopf-1 (DKK1) is a secreted protein which is known to promote the proliferation of various cancer cells, but its molecular mechanism has been unclear for many years. In 2016, Professor Kikuchi's group identified Cytoskeleton- Associated Protein 4 (CKAP4) as the cell membrane receptor for DKK1. When DKK1 binds to CKAP4, the PI3 kinase-AKT pathway is activated to promote cancer cell proliferation. Professor Kikuchi's group has reported that the DKK1-CKAP4 signaling pathway is activated in pancreatic, esophageal, liver, and lung cancers, and correlates with poor prognosis. They have also shown that the anti-CKAP4 antibody has anti-tumor effects using mouse models of these cancers. Additionally, they have discovered that CKAP4 on the plasma membrane is released into the extracellular space with exosomes and can be detected in human serum using ELISA. For instance, the serum CKAP4 level in unresectable pancreatic cancer cases was higher than in resectable patients or healthy individuals, and decreases significantly after surgical resection of the primary lesion. In this study, the group investigated new insights into the molecular mechanism of CKAP4 secretion via exosomes and the clinical significance of the serum CKAP4 level using 92 cases of lung cancer. In addition, they evaluated a novel lung cancer treatment strategy using the anti-CKAP4 antibody.
Research Results
1. DKK1 and CKAP4 proteins were highly expressed in A549 and Calu-1 cells among eight lung cancer cell lines, (CKAP4 was confirmed to present on the plasma membrane to function as a cell surface receptor). CKAP4 was detected in exosome fractions from the culture supernatants of A549 and Calu-1 cells. When clathrin, Rab27, and VPS35, which are involved in exosomal biogenesis, were knocked down in these cells, exosomal CKAP4 was decreased. Furthermore, when CKAP4 was overexpressed in NCI-H292 cells that do not express CKAP4 on the plasma membrane (NCI-H292/CKAP4 cells), CKAP4 was found to be present on the plasma membrane and secreted with exosomes. Thus, it was revealed that CKAP4 is secreted with exosomes in lung cancer cells, similar to pancreatic cancer cells.
2. In a collaborative study with Professor Yasushi Shintani, Professor Eiichi Morii (Graduate School of Medicine, Osaka University), and Professor Eiji Miyoshi (Graduate School of Health Sciences, Osaka University), the expression of DKK1 and CKAP4 in 92 cases of lung cancer was immunohistochemically analyzed, and the expression level was compared with clinicopathological data. The CKAP4 levels in the patient’s serum were measured using an ELISA assay using two different anti-CKAP4 antibodies. Among 92 cases, cases which expressed both DKK1 and CKAP4 showed poor prognosis, larger tumor size, and deeper invasion than cases expressing either one or neither. Furthermore, when serum CKAP4 level in lung cancer patients was compared with that of serum samples from matched healthy individuals (matched for age and sex), 19.6% of lung cancer patients but only 6.5% of healthy individuals had positive CKAP4 levels (> 0.1 ng/ml). In CKAP4-immunohistochemical positive cases, 24.3% had positive CKAP4 levels in the serum, while only 4.5% of CKAP4-immunohistochemical negative cases showed positive CKAP4 levels. In eight lung cancer cases in which serum CKAP4 levels were measured pre- and post- surgery, the levels of all cases decreased after surgery. Therefore, the results indicate that the serum CKAP4 level reflects CKAP4 expression in lung cancer lesions.
3. CKAP4 is localized in lipid rafts on the plasma membrane via palmitoylation of cytoplasmic cysteine residues at amino acid number 100 (Cys100). The CKAP4 mutant in which Cys100 of the palmitoylation site was mutated to serine (CKAP4C100S), which is not palmitoylated, exhibited the decreased secretion into the extracellular space via exosome. By analyzing vesicular transport via endosomal pathway using the novel Magnetic Activated Cell Sorting (MACS) system, it was shown that palmitoylation of CKAP4 enhances its traffic to the extracellular vesicle than lysosome from the multivesicular body.
4. Overexpression of CKAP4 in NCI-H292 cells (NCI-H292/CKAP4 cells) resulted in increased AKT activity and enhanced cell proliferation both in vivo and in vitro experiments. Treatment with anti-CKAP4 monoclonal antibody inhibited cell proliferation of NCI-H292/CKAP4 cells both in vivo and in vitro experiments. Furthermore, serum CKAP4 of mice transplanted with NCI-H292/CKAP4 cells was elevated, which was significantly reduced by the administration of anti-CKAP4 antibody.
5. EGFR mutations are observed in about 50% of non-small cell lung cancer patients in Asian countries including Japan, and EGFR tyrosine kinase inhibitors (EGFR-TKIs) have improved prognosis of these patients. The third-generation EGFR-TKI, osimertinib, is currently one of the standard therapeutics for the patients with EGFR mutation, but the emergence of resistant cells has become a problem. Expression of DKK1 and CKAP4 was evaluated in five lung cancer cell lines with EGFR mutations, and HCC-4006 cells highly expressed both proteins. Monotherapy with either the anti-CKAP4 antibody or osimertinib inhibited AKT activity and cell proliferation of HCC-4006 cells both in vivo andin vitro experiments. Furthermore, the combination of both drugs exhibited a synergistic therapeutic effect to inhibit tumor cell proliferation in vitro and in vivo models.
Significance of Research Findings
As a conclusion of this study, both DKK1 and CKAP4 proteins were found to be highly expressed in 41 cases (44.6%) out of 92 cases of lung cancer, and these cases exhibited severe malignancy and poor prognosis. These results are consistent with the previous reports from Professor Kikuchi’s lab on pancreatic cancer, esophageal cancer, and liver cancer. In addition, the serum CKAP4 level was found to be higher in lung cancer patients than in healthy individuals, and a significant decrease in the serum CKAP4 level was observed after surgical resection of primary disease. Therefore, it is suggested that the serum CKAP4 level could be applicable for a tumor marker and a prognosis marker. Furthermore, a new treatment strategy using anti-CKAP4 antibody in combination with osimertinib was proposed, which exhibited a synergistic anti-tumor effect. Lung cancer is a disease with high mortality and mortality rates worldwide, so the development of novel drugs is in critical need. It is expected that this research will contribute to the development of a new therapeutic strategy.
The article, “CKAP4 is a potential exosomal biomarker and a therapeutic target for lung cancer” was published in Transl. Lung Cancer Res. on March 17th, 2023.
【Title】
CKAP4 is a potential exosomal biomarker and a therapeutic target for lung cancer
【Journal】
Translational Lung Cancer Research.
【Authors】
Akihiro Nagoya, Ryota Sada, Hirokazu Kimura, Hideki Yamamoto, Koichi Morishita, Eiji Miyoshi, Eiichi Morii, Yasushi Shintani, and Akira Kikuchi
Supported by:
Grants-in-Aid for Scientific Research (S) and the AMED Next-Generation Cancer Creation Strategy.
