Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma

PLOS ONE RESEARCH ARTICLE Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma Nontawat Benjakul ID1,2, Nattapa Prakobphol3, Chayada Tangshewinsirikul4☯, Wirada Dulyaphat ID4☯, Jisnuson Svasti3,5, Komgrid Charngkaew1‡, Thaned Kangsamaksin ID3‡* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Faculty of Medicine Siriraj Hospital, Department of Pathology, Mahidol University, Bangkok, Thailand, 2 Faculty of Medicine Vajira Hospital, Department of Anatomical Pathology, Navamindradhiraj University, Bangkok, Thailand, 3 Faculty of Science, Department of Biochemistry, Mahidol University, Bangkok, Thailand, 4 Faculty of Medicine Ramathibodi Hospital, Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Mahidol University, Bangkok, Thailand, 5 Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * OPEN ACCESS Citation: Benjakul N, Prakobphol N, Tangshewinsirikul C, Dulyaphat W, Svasti J, Charngkaew K, et al. (2022) Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma. PLoS ONE 17(12): e0279001. https://doi.org/ 10.1371/journal.pone.0279001 Editor: Shuai Ren, Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine, CHINA Received: August 18, 2022 Accepted: November 28, 2022 Published: December 22, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0279001 Copyright: © 2022 Benjakul 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. Abstract Vasculogenic mimicry (VM) is the process where cancer cells adopt endothelial characteristics by forming tube-like structures and perfusing channels. This phenomenon has been demonstrated in several types of solid tumors and associated with the growth and survival of tumor cells. In this study, we investigated the presence of VM formation in human pancreatic ductal adenocarcinoma (PDAC) and elucidated the molecular mechanisms underlying the VM process. In human PDAC tissues, CD31-negative, periodic acid-Schiff (PAS)-positive channels were predominantly found in desmoplastic areas, which are generally also hypovascularized. We found a positive correlation of VM capacity to tumor size and NOTCH1 expression and nuclear localization with statistical significance, implicating that Notch activity is involved with VM formation. Additionally, our data showed that the presence of growth or angiogenic factors significantly increased Notch activity in PDAC cell lines and upregulated several mesenchymal marker genes, such as TWIST1 and SNAI1, which can be inhibited by a gamma-secretase inhibitor. Our data showed that Notch signaling plays an important role in inducing VM formation in PDAC by promoting the epithelial-to-mesenchymal transition process. Introduction Pancreatic cancer is among the most lethal human cancer with an average 5-year survival rate of less than 5% [1]. There are a number of types of pancreatic cancer, but pancreatic ductal PLOS ONE | https://doi.org/10.1371/journal.pone.0279001 December 22, 2022 1 / 15 PLOS ONE Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research has received funding support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (Grant Number B05F640133), National Research Council of Thailand and Mahidol University (Grant Number NRCT5-RSA63015-11) (T.K.) and the Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University (K.C.). The details of the funders can be found at (1) https://www.nxpo.or.th/B, (2) https://www.nrct.go. th, and (3) https://www.mahidol.ac.th. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Notch signaling regulates vasculogenic mimicry in pancreatic cancer adenocarcinoma (PDAC) is the most common and accounts for about 85% of cases. Surgical resection is the only cure, which can improve the 5-year survival rate up to 20% [2]. However, most pancreatic cancer patients are often diagnosed at an advanced stage, which renders treatable surgery impossible. In addition, pancreatic cancer has been shown to be insensitive to many chemotherapeutic drugs. The current standard-of-care therapy improves patient survival by only a matter of weeks [3]. It is widely accepted that the growth of solid tumors requires a continuous supply of oxygen and nutrients [3,4]. One of the most important factors and characteristic hallmarks for cancer development and progression is the angiogenic switch. Therefore, genetic manipulations and pharmacological perturbations of angiogenesis have been extensively investigated, and data from various studies have led to the development of a number of cancer therapeutics targeting angiogenic processes [5]. Bevacizumab (Avastin, Genentech/Roche), one of the first clinicallyapproved angiogenesis inhibitors, has been used to treat patients with metastatic colorectal, lung, renal, and ovarian cancers by blocking the vascular endothelial growth factor (VEGF) pathway [6]. However, after the initial response phase from anti-angiogenic treatment, tumors often begin to resurface and the disease progression resumes. Increasing evidence demonstrates that tumor cells develop an adaptive response and become resistant to angiogenesis inhibitors. There are several proposed mechanisms of how a tumor can evade angiogenesis inhibition, including upregulation of alternative pro-angiogenic signaling pathways, increased protection of tumor vasculature from anti-angiogenic drugs, and vasculogenic mimicry [5–8]. Vasculogenic mimicry (VM) has been characterized by the ability of cancer cells to express endothelium-associated genes and generate extracellular matrix (ECM)-rich vascular networks, which recapitulate embryonic vasculogenesis. This process has been associated with human aggressive tumors, including metastatic melanoma cells [9]. VM has been observed in other types of cancer, such as carcinomas (breast, ovary, lung, prostate, bladder, kidney); sarcomas (Ewing, mesothelial, (...truncated)