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DEAD-Box Helicase 27 Enhances Stem Cell-Like Properties with Poor Prognosis in Breast Cancer

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DEAD-Box Helicase 27 Enhances Stem Cell-Like Properties with Poor Prognosis in Breast Cancer DEAD-Box Helicase 27 Enhances Stem Cell-Like Properties With Poor Prognosis in Breast Cancer Shan Li The First Aliated Hospital of China Medical University Jinfei Ma The First Aliated Hospital of China Medical University Ang Zheng The First Aliated Hospital of China Medical University Xinyue Song China Medical University Si Chen China Medical University Feng Jin ( [email protected] ) The First Aliated Hospital of China Medical University https://orcid.org/0000-0002-0325-5362 Research Article Keywords: DEAD-box helicase 27 (DDX27), Breast cancer, Stem cell-like properties, Prognosis Posted Date: June 7th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-521379/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Journal of Translational Medicine on August 6th, 2021. See the published version at https://doi.org/10.1186/s12967-021-03011-0. Page 1/22 Abstract Background Although the rapid development of diagnosis and treatment has improved prognosis in early breast cancer, challenges from different therapy response remain due to breast cancer heterogeneity. DEAD-box helicase 27 (DDX27) had been proved to inuence ribosome biogenesis and identied as a promoter in gastric and colorectal cancer associated with stem cell-like properties, while the impact of DDX27 on breast cancer prognosis and biological functions is unclear. We aimed to explore the inuence of DDX27 on stem cell-like properties and prognosis in breast cancer. Methods The expression of DDX27 was evaluated in 24 pairs of fresh breast cancer and normal tissue by western blot. We conducted Immunohistochemical (IHC) staining in paran sections of 165 breast cancer patients to analyze the expression of DDX27 and its correlation to stemness biomarker. The Cancer Genome Atlas-Brest Cancer (TCGA-BRCA) and Kaplan-Meier survival analysis were used to investigate the expression of DDX27 in breast cancer and its implication on prognosis. Western blot, CCK-8 assay, Transwell assay and wound-healing assay were used to clarify the regulation of DDX27 on stem cell-like properties in breast cancer cells. Gene Set Enrichment Analysis (GSEA) was performed to analyze the potential molecular mechanisms of DDX27 in breast cancer. Results DDX27 was signicantly high expressed in breast cancer compared with normal tissue. High expression of DDX27 was related to larger tumor size (p = 0.0005), positive lymph nodes (p = 0.0008), higher histological grade (p = 0.0040), higher ki-67 (p = 0.0063) and later TNM stage (p 0.0001). Patients with high DDX27 expression turned out worse prognosis on overall survival (OS, p = 0.0087) and disease-free survival (DFS, p = 0.0235). Overexpression of DDX27 could enhance the expression of biomarkers related to stemness and promote stem cell-like activities such as proliferation and migration in breast cancer cells. Conclusion DDX27 can enhance stem cell-like properties and cause poor prognosis in breast cancer, also may be expected to become a potential biomarker for breast cancer therapy. Background Page 2/22 Breast cancer has already surpassed lung cancer and developed into the major malignant tumor in women all around the world with 2.3 million new cases in 2020 based on GLOBOCAN 2020[1]. Although the rapid development of diagnosis and treatment has improved prognosis in early breast cancer, challenges from different therapy response remain due to breast cancer heterogeneity. DEAD-box helicase 27 (DDX27) pertains to the DEAD-box RNA helicases family, which is a classical ATP- dependent helicases family containing conserved D-E-A-D (Asp-Glu-Ala-Asp) sequences. This family has been conrmed to participate in various processes containing RNA transportation, RNA degradation, glucose metabolism, lipid metabolism, ribosome biosynthesis, tumorigenesis, cancer development and so on[2–8]. DDX27 was proved to take part in the processes of ribosome biogenesis, which performed an important function in cell proliferating. DDX27 could regulate the 47S ribosome RNA formation and associated with PeBow-complex independently[9]. In the process of skeletal muscle myogenesis, DDX27 was reported to inuence ribosome RNA maturation, ribosome biogenesis and specic transcription[10]. DDX27 was also related to the development of malignant tumor. DDX27 had been conrmed to promote the development and metastasis in hepatocellular carcinoma and gastrointestinal cancer with poor prognosis[11–13]. Until now, the status of DDX27 expression and its implication on breast cancer remains unclear. Breast cancer stem cells refer to a fraction of cells who has strong self-renewal capacity and a huge potential of multiple differentiation in breast cancer which can promote the courses of tumorigenesis, development, metastasis and drug resistance[14–16]. Studies proved that members of DEAD-box RNA helicases family could affect the biological behavior of cancer stem cells in various cancers[17–19]. The only member of DEAD-box RNA helicases family which has exactly effects on breast cancer stem cells is DDX17, who can enhance stem cell-like activities by combining with SOX2 or mechanism of promoting stem-like properties under hypoxia[20, 21]. DDX27 was reported to promote stem cell-like characteristics and cause poor prognosis in gastric and colorectal cancer[11, 22], while the inuence on stemness in breast cancer remains unclear. Hence, it is of great signicance to explore the inuence of DDX27 on tumorigenesis, progress and the association with stem cell-like properties in breast cancer, which might suggest a new idea for diagnosis and therapy. In our study, DDX27 was highly expressed in both The Cancer Genome Atlas-Brest Cancer (TCGA-BRCA) database and breast cancer samples. High expression of DDX27 was rstly reported in breast cancer with the connection to clinicopathological factors and caused a shorter survival. The expression of DDX27 was positively related to stemness biomarkers and promoted the stem cell-like activities. Furthermore, Gene Set Enrichment Analysis (GSEA) suggested that DDX27 might have an effect on breast cancer by various ways. Hence, DDX27 can enhance the stem cell-like properties meanwhile leading to a poor prognosis in breast cancer, which means DDX27 may become a potentially signicant prognosis biomarker and therapeutic target. Methods Page 3/22 The Cancer Genome Atlas-Brest Cancer Database All of the gene expression data were downloaded and organized from TCGA-BRCA database (https://cancergenome.nih.gov/), which contains 1109 breast cancer and 103 normal samples. Normalization of all data was performed by edgR package. Kaplan-Meier survival analysis and Gene Set Enrichment Analysis Kaplan-Meier Plotter (http://kmplot.com/analysis/) is an online database containing survival information and gene expression of various cancers and used to analyze the implication of DDX27 on breast cancer prognosis [23]. The potential mechanism of DDX27 expression in breast cancer was analyzed by GSEA 4.0.3 software. Patients and tissue samples Under the permission from the Ethics Review Committee in the First Aliated Hospital of China Medical University (AF - SOP − 07–1.1–01), fresh cancer tissue with paired adjacent normal tissue (n = 24), and paran-embedded specimens (n = 165) were obtained. All included patients were pathologically diagnosed as inltrative ductal carcinoma. None of the patients received breast-conserving surgery or neoadjuvant therapy. Twenty-four pairs of fresh specimens were collected within 30 minutes after surgery and 165 paran-embedded specimens for IHC were collected during Jan.2014 to Dec.2015. Clinical and pathological information obtained from the Hospital Information System included age, tumor size, lymph nodes status, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (HER2) status, histological grade, ki-67 index and TNM stage. Western blot Samples of tissue and cells were lysed by the compound of RIPA buffer and protease inhibitors (Sigma- Aldrich) for half an hour on ice after washed by phosphate-buffered saline (PBS) twice. Then the samples were centrifuged for 15 minutes in the condition of 14,000×g and 4°C. Protein from tissue and cells was quantied by BCA assay kit (Beyotime, Jiangsu, China). Protein with equal quantity was transferred onto the polyvinylidene uoride (PVDF) membranes (Millipore, Bedford, MA) after electrophoresed by SDS- PAGE. The membrane was sealed with bovine serum albumin (BSA) and hatched with primary antibodies at 4℃ overnight, then the secondary antibodies at room temperature for one hour. The primary antibodies for experiments contained anti-DDX27 (1:2000, Abcam, USA), anti-SOX2 (1:1000, Proteintech, China), anti-OCT4 (1:1000, Cell Signaling Technology, USA) and β-actin (1:2000, Proteintech, China). Membranes were tested with an enhanced chemiluminescence detection kit (BOSTER, USA). Immunohistochemical staining Each paran-embedded sample was cut into 4µm sections, dewaxed routinely, and dehydrated with gradient ethanol. All sections were retrieved antigen in citrate buffer with high pressure. The sections were restored to room temperature and blocked the activity of endogenous peroxidase by 3% hydrogen Page 4/22 peroxide. Then, all the slides were incubated with anti-DDX27 (1:2000, Novus Biologicals, USA) or anti- OCT4 (1:500, Cell Signaling Technology, USA) at 4°C overnight. The sections were incubated with the secondary antibody at room temperature
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