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Human Ribonuclease 1 Serves As a Secretory Ligand of Ephrin A4 Receptor and Induces Breast Tumor Initiation

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Human Ribonuclease 1 Serves As a Secretory Ligand of Ephrin A4 Receptor and Induces Breast Tumor Initiation ARTICLE https://doi.org/10.1038/s41467-021-23075-2 OPEN Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation Heng-Huan Lee 1,16, Ying-Nai Wang 1,16, Wen-Hao Yang 1,2,16, Weiya Xia1, Yongkun Wei1, Li-Chuan Chan1, Yu-Han Wang1,2, Zhou Jiang 1, Shouping Xu3, Jun Yao1, Yufan Qiu1,4, Yi-Hsin Hsu1, Wei-Lun Hwang5,6, Meisi Yan1,7, Jong-Ho Cha 1,8, Jennifer L. Hsu1, Jia Shen1, Yuanqing Ye9, Xifeng Wu9, Ming-Feng Hou10,11, Lin-Ming Tseng12,13, Shao-Chun Wang 2, Mei-Ren Pan11, Chin-Hua Yang12, Yuan-Liang Wang2, ✉ Hirohito Yamaguchi1,2, Da Pang3, Gabriel N. Hortobagyi 14, Dihua Yu1 & Mien-Chie Hung 2,1,15 1234567890():,; Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonu- cleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis. 1 Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 2 Graduate Institute of Biomedical Sciences, Research Center for Cancer Biology, and Center for Molecular Medicine, China Medical University, Taichung, Taiwan. 3 Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin, China. 4 Deptartment III of Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. 5 Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Hsinchu, Taiwan. 6 Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan. 7 Department of Pathology, Harbin Medical University, Harbin, China. 8 Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, Korea. 9 Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 10 Division of Breast Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. 11 Graduate Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 12 Comprehensive Breast Health Center and Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan. 13 Institute of Clinical Medicine and Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 14 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 15 Department of Biotechnology, Asia University, Taichung, Taiwan. 16These authors contributed equally: Heng- ✉ Huan Lee, Ying-Nai Wang, Wen-Hao Yang. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2788 | https://doi.org/10.1038/s41467-021-23075-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23075-2 he pancreatic hRNase A superfamily is comprised of eight negative correlation with breast cancer patient survival, but not canonical hRNases, including human ribonuclease 1 that of the other seven hRNases, including hRNases 2, 3, 4, 5, 6, 7, T fi (hRNase 1), and ve noncanonical hRNases, all of which and 11 (Supplementary Fig. 1a, b). Of note, the survival analysis can be detected in human plasma/serum1,2. Secretory hRNase 1 is was based on the database of Kaplan-Meier Plotter, in which the found in various organs3,4, and its biochemical properties and microarray contains 22,277 genes detected for breast cancer post-translational modifications, such as glycosylation, have been prognosis, but RNases 8, 9, and 10 are not included in the extensively investigated5–7. Nonetheless, the biological function of analysis35. Moreover, higher hRNase 1 expression significantly hRNase 1 has not yet been completely defined. Studies on bovine correlated with poorer survival in tumors with grade 2 in a near- homolog RNase A indicated that its human homolog hRNase 1 significant trend (Fig. 1b), and hRNase 1 appeared to be a more also plays a role in the digestive system by degrading dietary significant poor prognostic factor in breast cancer patients with RNAs4,8. Moreover, hRNase 1 regulates hemostasis, inflamma- lymph node (LN)-negative non-metastasis, compared with those tion, and innate immunity2, indicating that hRNase 1 possesses with LN-positive metastasis (Fig. 1c). Among the four molecular multiple functions in addition to RNA clearance9–12. Recent subtypes of breast cancer36, hRNase 1 correlated negatively with studies indicated that hRNase 5 serves as a ligand for the receptor the survival of breast cancer patients with the basal-like, luminal tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) A and luminal B subtypes, in a trend toward statistical sig- and plexin-B2 (PLXNB2) receptor13–15 in solid and hemato- nificance (Supplementary Fig. 2a). Interestingly, the luminal B poietic cancers. Together, those findings reveal an unconventional subtype in each grade displayed a higher hazard ratio and a ligand–receptor relationship and a role of the hRNase A super- smaller p value, suggesting a stronger correlation between high family in tumor progression16–18. hRNase 1 expression and poor patient survival in the luminal B The family of erythropoietin-producing hepatocellular carci- subtype, although there is a lack of statistical significance (Sup- noma (Eph) receptors is the largest among the RTKs with plementary Fig. 2b). In addition, hRNase 1 expression predicted important functions in embryonic development and adult tissue poorer outcome in LN-positive patients with the luminal A homeostasis19. Eph receptors are traditionally thought to bind to subtype and LN-negative patients with the luminal B subtype their membrane-bound ligands, ephrins, on neighboring cells to (Supplementary Fig. 2c). The notion that higher hRNase 1 stimulate juxtacrine signals through contact-dependent cell–cell expression associated with poorer patient survival was also communication20. Interestingly, soluble ephrin-A ligands derived demonstrated in two other independent databases of patients from their membrane-bound forms have been detected in the with breast cancer37,38 (Supplementary Fig. 3a, b). culture media of cell lines in models of glioblastoma, breast and To further investigate the role of hRNase 1 in breast cancer, we other cancers, where they activate EphA receptors and down- examined tumor histology using a breast tumor tissue microarray stream signaling, and cellular responses21–24. It is noteworthy that (TMA) and found that the average expression level of hRNase 1 metalloprotease-mediated proteolytic shedding of membrane- in breast tumors (T) was significantly higher than that in normal anchored ephrins has been further demonstrated as one major (N) breast tissues (Fig. 1d). Of note, the levels of tissue hRNase 1 mechanism to generate soluble ephrins in the extracellular had no statistically significant difference among the four subtypes space25,26. The potential function of Eph receptors in human of breast cancer (Fig. 1e). Consistently, serum hRNase 1 level was cancers, including breast cancer, suggest that they can be targeted significantly higher in breast cancer patient group (T) compared for cancer treatment27–29. Notably, ephrin A4 (EphA4) is upre- with that in normal group (N) (Fig. 1f), which was also observed gulated in breast cancer stem-like cells (CSCs), and EphA4- in three other independent cohorts (Supplementary Fig. 4a–c), mediated juxtacrine signaling maintains the stem cell state and shown no significant difference among breast cancer through their interactions with tumor-associated monocytes/ subtypes (Fig. 1g and Supplementary Fig. 4d, e). Likewise, the macrophages and CSCs30. CSC-like cells have been shown to average expression level of the conditioned medium (CM) contribute to tumorigenicity, invasiveness, and immune evasion, hRNase 1 in eight breast cancer cell lines was significantly higher and are ideal targets in cancer treatment31–33. In addition to the than that in two normal breast cells (Fig. 1h). Further analysis classical juxtacrine signaling, further investigations into the indicated that these eight breast cancer cell lines were determined autocrine/paracrine signaling involved in EphA4 activation may at a concentration of CM hRNase 1 lower than 15 ng/ml, except provide a more comprehensive picture of Eph receptor regulation KPL4 cells carrying CM hRNase 1 up to 100 ng/ml (Supplemen- and shed light on potential therapeutic strategies against CSC-like tary Fig. 4f). We further performed western blotting (WB) to cells by blocking
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