CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma

PLOS ONE RESEARCH ARTICLE CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma Chien-Liang Liu1, Hung-Wen Tsai2, Shu-Ling Peng2, Ching-Ping Chang ID3, YuHao Chang ID4, Huei-Sheng Huang ID4* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Division of Urology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan, 2 Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 3 Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan, 4 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan * Abstract OPEN ACCESS Citation: Liu C-L, Tsai H-W, Peng S-L, Chang C-P, Chang Y-H, Huang H-S (2023) CDCP1 (CUB domain containing protein 1) is a potential urinebased biomarker in the diagnosis of low-grade urothelial carcinoma. PLoS ONE 18(3): e0281873. https://doi.org/10.1371/journal.pone.0281873 Editor: Tsai-Ching Hsu, Chung Shan Medical University, TAIWAN Received: October 19, 2022 Accepted: February 1, 2023 Published: March 2, 2023 Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: HSH This work was supported by grants from the Ministry of Science and Technology (Taipei, Taiwan; MOST104-2314-B-006-055-MY3, MOST107-2314-B-006-019-MY3), and Chi Mei Medical Center (Tainan, Taiwan; CMNCKU10814). No. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Urine-based cytology is non-invasive and widely used for clinical diagnosis of urothelial carcinoma (UC), but its sensitivity is less than 40% for low-grade UC detection. As such, there is a need for new diagnostic and prognostic biomarkers of UC. CUB domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein highly expressed in various cancers. Using tissue array analysis, we demonstrated that CDCP1 expression in UC patients (n = 133), especially in those with low-grade UC, was significantly higher than in 16 normal persons. In addition, CDCP1 expression in urinary UC cells could also be detected by using immunocytochemistry method (n = 11). Furthermore, in 5637-CD cells, overexpression of CDCP1 affected the expression of epithelial mesenchymal transition-related markers and increased matrix metalloproteinase 2 expression and migration ability. Conversely, the knockdown of CDCP1 in T24 cells had the opposite effects. Using specific inhibitors, we demonstrated the involvement of c-Src/PKCδ signaling in the CDCP1-regulated migration of UC. In conclusion, our data suggest that CDCP1 contributes to the malignant progression of UC and may have the potential as a urine-based biomarker for detecting low-grade UC. However, a cohort study needs to be conducted. Introduction Urothelial carcinoma (UC) of the bladder is estimated to be the 4th commonly diagnosed cancer and ranked 8th in cancer-related death in males in the United States in 2022 [1]. Some risk factors for the high prevalence of UC worldwide include tobacco smoking and ingestion of arsenic contaminated water or herbal medicines containing aristolochic acid. However, the exact mechanism of the disease is not yet fully elucidated. The high recurrence rate of UC causes the need for long-term surveillance by regular cytologic and cystoscopy examinations for follow-up UC patients [2]. Presently, cystoscopy or a combination of urinary cytology is the gold standard for UC diagnosis. However, cystoscopy is PLOS ONE | https://doi.org/10.1371/journal.pone.0281873 March 2, 2023 1 / 15 PLOS ONE Competing interests: The authors have declared that no competing interests exist. Abbreviations: UC, Urothelial carcinoma; CDCP1, CUB domain-containing protein 1; IHC, Immunohistochemistry; ICC, Immunocytochemistry; SFK, Src family kinase; RTKs, Receptor tyrosine kinases; FCS, Fetal calf serum; PVDF, Polyvinylidene difluoride; MMPs, Metalloproteases; N/C, Nuclear/Cytoplasmic ratio. CDCP1 as a potential biomarker to detect low-grade UC unsuitable for screening UC patients because the frequently invasive operation might make patients uncomfortable and incur high medical costs. The major advantages of urinary cytology with the Papanicolaou (PAP) stain method are its non-invasiveness, lower turnaround time in the laboratory, and cost-effectiveness, and has been widely used as a screening or a follow-up tool for patients not only in UC but also in cervical cancer [3, 4]. One disadvantage of the PAP stain method is the requirement of a well-trained cytopathologist to clarify malignant cells according to their morphological features, such as a high nuclear/cytoplasmic ratio (N/ C), aggregating clusters, increased cellularity, irregular nuclear margins, hyperchromasia, and chromatin abnormality [5]. Nonetheless, there are still no standard criteria for the morphological evaluation, especially in an atypical status, which often confuses experienced cytopathologists [6, 7]. Further, preserved or inadequate urine specimens, clinical conditions, and laboratory processing also influence the accuracy and quality of cytology. Therefore, urinary cytology with the PAP stain method is suitable for high-grade UC diagnosis and prognosis; however, its low sensitivity for detecting low-grade UC is a concern [8, 9]. In brief, finding new diagnostic and prognostic biomarkers of UC is eagerly needed to improve the sensitivity of urinary cytology. CUB domain-containing protein 1 (CDCP1) is a type I transmembrane glycoprotein. Fulllength CDCP1 (135–140 kDa) comprises three domains. The extracellular domain can be cleaved at the R368 and K369 by some proteases, such as matriptase, plasmin, trypsin, and urokinase, generating a smaller C-terminal transmembrane fragment (75–85 kDa) [10, 11]. The CUB (complement C1r/C1s, Uegf, and Bmp1) domain has been suggested to play an important role in the developmental process [12]. It also plays a critical role in tumor metastasis to regulate anoikis resistance in lung cancer cells [13]. It promotes pancreatic cancer migration, invasion, and extracellular matrix (ECM) degradation in a phosphorylation-dependent manner [14]. CDCP1 can mainly serve as a substrate for the binding of Src family kinase (SFK), including Src, Fyn, and Yes, at Y734 of the cytoplasmic domain to recruit PKCδ to the membrane and interact with Y762 site to induce anoikis resistance, cell migration, and extracellular matrix degradation [13]. It can also interact with other receptor tyrosine kinases and cellular surface proteins as a hub to relay si (...truncated)