IGFBP5 is an ROR1 ligand promoting glioblastoma invasion via ROR1/HER2-CREB signaling axis

Article https://doi.org/10.1038/s41467-023-37306-1 IGFBP5 is an ROR1 ligand promoting glioblastoma invasion via ROR1/HER2-CREB signaling axis Received: 13 December 2021 Check for updates 1234567890():,; 1234567890():,; Accepted: 10 March 2023 Weiwei Lin1,2,3,4,8, Rui Niu1,8, Seong-Min Park2,5,8, Yan Zou 1,6,8, Sung Soo Kim 2,8, Xue Xia1, Songge Xing1, Qingshan Yang1, Xinhong Sun1, Zheng Yuan1, Shuchang Zhou1, Dongya Zhang1, Hyung Joon Kwon7, Saewhan Park2, Chan Il Kim 2, Harim Koo2, Yang Liu1, Haigang Wu1, Meng Zheng1, Heon Yoo2,3, Bingyang Shi 1,6 , Jong Bae Park 1,2,3 & Jinlong Yin 1,2 Diffuse infiltration is the main reason for therapeutic resistance and recurrence in glioblastoma (GBM). However, potential targeted therapies for GBM stem-like cell (GSC) which is responsible for GBM invasion are limited. Herein, we report Insulin-like Growth Factor-Binding Protein 5 (IGFBP5) is a ligand for Receptor tyrosine kinase like Orphan Receptor 1 (ROR1), as a promising target for GSC invasion. Using a GSC-derived brain tumor model, GSCs were characterized into invasive or non-invasive subtypes, and RNA sequencing analysis revealed that IGFBP5 was differentially expressed between these two subtypes. GSC invasion capacity was inhibited by IGFBP5 knockdown and enhanced by IGFBP5 overexpression both in vitro and in vivo, particularly in a patient-derived xenograft model. IGFBP5 binds to ROR1 and facilitates ROR1/HER2 heterodimer formation, followed by inducing CREB-mediated ETV5 and FBXW9 expression, thereby promoting GSC invasion and tumorigenesis. Importantly, using a tumor-specific targeting and penetrating nanocapsule-mediated delivery of CRISPR/Cas9-based IGFBP5 gene editing significantly suppressed GSC invasion and downstream gene expression, and prolonged the survival of orthotopic tumor-bearing mice. Collectively, our data reveal that IGFBP5-ROR1/HER2-CREB signaling axis as a potential GBM therapeutic target. Patients with glioblastoma (GBM), the most frequent and aggressive malignant primary brain tumor in adults, have an average overall survival time of merely 14 months1,2. The infiltrative nature of GBM enables neoplastic spread and migration into adjacent brain tissue, which makes it very challenging, or even impossible, for all multimodality treatments to achieve complete excision, inevitably leading to recurrence3–5. Therefore, elucidating the mechanisms regulating GBM invasion is key for the development of effective therapeutic strategies. 1 Henan-Macquarie University Joint Centre for Biomedical Innovation, School of Life Sciences, Henan University, Kaifeng, Henan 475004, China. 2Department of Cancer Biomedical Science, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Gyeonggi 10408, Republic of Korea. 3 Research Institute, National Cancer Center, Goyang, Gyeonggi 10408, Republic of Korea. 4Department of Life Science, Ewha Womans University, Seoul 03760, Republic of Korea. 5Personalized Genomic Medicine Research Center, KRIBB, Daejeon 34141, Republic of Korea. 6Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, NSW 2109, Australia. 7Department of Cancer Control and Population Health, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Gyeonggi 10408, Republic of Korea. 8These e-mail: ; ; authors contributed equally: Weiwei Lin, Rui Niu, Seong-Min Park, Yan Zou, Sung Soo Kim. Nature Communications | (2023)14:1578 1 Article GBM stem-like cells (GSCs), also known as GBM-initiating cells, are responsible for disease progression, therapeutic resistance, and tumor recurrence6–8. These cells share stem cell markers with neural stem cells, such as Nestin and CD133, as well as their capacity of self-renewal and differentiation5,9,10. In contrast to differentiated tumor cells, GSCs can efficiently propagate tumors in orthotopic xenograft mice6,9,11. Moreover, GSC-derived orthotopic xenografts closely mirror the phenotype and genotype of primary tumors in patients11. In this xenograft mouse model, both invasive and localized orthotopic tumors are established from a series of human GSCs12–16. The exhibition of various degrees of invasive model promptly resembles the brain pathological features of patients with GBM, is ideal for the thorough investigation of the molecular mechanism underpinning GBM invasion. Recently, a comprehensive longitudinal study of GBM tumors classified GBMs into proneural (PN), classical (CL), and mesenchymal (MES) subtypes, and a similar recapitulation can be made for GSCs13,17. In particular, according to the Ivy GAP transcriptome data (Ivy Glioblastoma Atlas Project), the PN subtype is predominantly documented in the leading edge of the tumor, as compared to the MES subtype which largely exists in the pseudopalisading region or the tumor core13,18. In addition, a recent phenotypic study of patients with GBM revealed that PN- and MES-subtyped GSCs were localized to the invasive edge and core of the tumor, respectively19. Nevertheless, the detailed molecular characteristics of these invasive and non-invasive GSCs remain largely unexplored. In this study, to identify the master regulators of invasive GSCs and to better understand GBM invasion, we perform RNA-seq analysis between invasive and non-invasive GSCs, which are divided according to the hematoxylin and eosin (H&E) staining of GSC-derived orthotopic xenograft models. We demonstrate that IGFBP5 regulates GSC invasion serving as a ligand for ROR1, which triggers formation of ROR1/HER2 (Human Epidermal growth factor Receptor 2) heterodimer to enhance CREB (cAMP Response Element Binding protein) oncogenic signaling. Moreover, both lentivirus-mediated IGFBP5 knockdown and nanocapsule-mediated Cas9/sgIGFBP5 delivery significantly compromise GSC invasion and extend the survival of orthotopic tumor-bearing mice. Collectively, our findings highlight the critical role of IGFBP5 in enhancing GSC invasion and providing a promising therapeutic approach for diffuse GBM. Results IGFBP5 expression is associated with GSCs invasion and patient survival in glioma To study GBM invasion, we classified GSCs into invasive or noninvasive by phenotypic characterization using orthotopic xenograft mouse models. In detail, 448 and X01 GSCs formed invasive tumors that spread into the brain through the corpus callosum, whereas 83 and 131 GSCs exhibited strong localization with a clear boundary, indicating non-invasive localization (Fig. 1a). In addition, in vitro transwell invasion assays demonstrated consistent results regarding the behavior of the two GSC subtypes: 448 and X01 GSCs exhibited significantly greater invasive ability than the non-invasive GSCs (83 and 131 GSCs; P < 0.01; Fig. 1b). High-throughput RNA sequencing (RNA-seq) was performed to further elucidate the molecular mechanism underlying the different invasive abilities of the two GSC types. Differentially expres (...truncated)