Published OnlineFirst July 6, 2018; DOI: 10.1158/1078-0432.CCR-18-0472 Cancer Therapy: Clinical Clinical Cancer Research Functions and Mechanisms of Tumor Necrosis Factor-a and Noncoding RNAs in Bone-Invasive Pituitary Adenomas Haibo Zhu1, Jing Guo1, Yutao Shen1, Wei Dong1, Hua Gao1, Yazhou Miao1, Chuzhong Li1,2,3,4, and Yazhuo Zhang1,2,3,4 Abstract Purpose: To explore the molecular mechanism and prog- pathways. Pathway act work showed that osteoclast differen- nosis of bone-invasive pituitary adenomas (BIPA). tiation pathway was significantly implicated in the pathway Experimental design: A total of 274 patients with pituitary network. BIPAs had higher expression of TNFa than that of adenomas were followed up. Transcriptomic microarrays NBIPAs on IHC. In vitro, TNFa could induce RAW264.7 cells to analysis was performed on 10 pituitary adenomas, including differentiate into mature osteoclasts, leading to bone destruc- five BIPAs and five non-bone-invasive pituitary adenomas tion. NR_033258, lncRNA SNHG24, miR-181c-5p, and miR- (NBIPA). The targeted molecular markers were validated by 454-3p can regulate TNFa expression. qRT-PCR, IHC, ELISA, and osteoclast differentiation. Conclusions: BIPAs had worse PFS than did NBIPAs in Results: Clinical variable analyses revealed a significant the NGTR and NFPA groups. Inflammatory and immune correlation between bone invasion and female sex, large factors play an important role in BIPAs. TNFa can directly tumor volume, non-gross total resection (NGTR), and tumor induce osteoclast differentiation in BIPAs. NR_033258, regrowth. BIPAs had worse progression-free survival (PFS) lncRNA SNHG24, miR-181c-5p, and miR-454-3p can reg- than did NBIPAs in the NGTR and nonfunctional pituitary ulate TNFa expression. TNFa anditsrelatedlncRNAs adenoma (NFPA) groups. Gene ontology functional and and miRNAs represent potential therapeutic targets for KEGG pathway analyses showed that the biological processes bone-invasive pituitary adenomas in the future. Clin Cancer and pathways were primarily immune and inflammatory Res; 1–10. Ó2018 AACR. Introduction analyze the prognosis, clinical features, and molecular mecha- nism of BIPAs to design and implement appropriate therapeutic Pituitary adenomas are benign tumors that originate in the interventions. anterior pituitary cells and account for approximately 8% to 15% Common diseases that lead to excessive bone destruction of intracranial tumors (1, 2). Large pituitary adenomas are typ- include tumors (bone metastases of cancer, osteosarcoma, giant ically invasive and infiltrate the surrounding dura, sinus, brain, cell tumors of the bone, neuroblastoma, chordoma, meningi- and bone tissue. Existing reports focus mainly on the suprasellar oma, etc.), periodontal disease (oral squamous cell carcinoma, and lateral-invasion of pituitary adenomas. There are few studies etc.), rheumatoid arthritis, and others (4–8). Tumor cells alone focused on the prognosis and mechanisms of bone-invasive cannot directly lead to bone destruction but can promote the pituitary adenomas (BIPA); most studies are clinical and imaging differentiation and maturation of osteoclasts to play a role in (3). Because of its serious destruction of the surrounding bone bone destruction. The differentiation and maturation of osteo- mass and large size, the surgical resection of BIPAs is a significant clasts is typically mediated by cellular components such as challenge and may increase the potential risk of cerebrospinal immune cells, the tumor cells and cytokines such as IL1/6, fluid leaks. Based on these factors, it is important to explore and TNFa,PTHrP,TGFb, CXCL13, CXCL12, etc. (9, 10). TNFa is a dominant cytokine that plays a critical role in the promotion of pathologic osteoclast formation leading to inflammatory bone 1Beijing Neurosurgical Institute, Capital Medical University, Beijing, China. destruction (11). In this study, we identified TNFa as a key 2Department of Neurosurgery, Beijing Tiantan Hospital affiliated to Capital 3 molecule involved in BIPAs. Medical University, Beijing, China. Beijing Institute for Brain Disorders Brain miRNAs are small, single-stranded, noncoding RNAs of Tumor Center, Beijing, China. 4China National Clinical Research Center for Neurological Diseases, Beijing, China. approximately 22 nucleotides that mediate homologous sequence-dependent gene silencing in cells. Research has dem- Note: Supplementary data for this article are available at Clinical Cancer onstrated that some miRNAs, such as miR-34c, miR-124, miR- Research Online (http://clincancerres.aacrjournals.org/). 223, etc., are involved in osteoclast differentiation (12–14). Long Corresponding Author: Yazhuo Zhang, Beijing Neurosurgical Institute, Beijing noncoding RNAs (lncRNA) are RNAs with transcript lengths of 100050, China. Phone: 86-10-67096763; E-mail: [email protected] >200 nucleotides that regulate gene expression through multiple doi: 10.1158/1078-0432.CCR-18-0472 mechanisms. A recent study showed that the lncRNA molecules Ó2018 American Association for Cancer Research. MAYA and lncRNA MALAT1 are involved in cancer cell–induced www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 6, 2018; DOI: 10.1158/1078-0432.CCR-18-0472 Zhu et al. January 1, 2008, to December 31, 2015. All patients were Translational Relevance classified according to preoperative images, including plain Bone-invasive pituitary adenomas (BIPA) are a rare cause of and enhanced head MRI, thin layer skull base CT scanning and pituitary adenomas, but their mechanisms and prognosis three-dimensional reconstruction. The patients were divided remain unknown. Because of its serious destruction of the into three groups: BIPAs, invasive pituitary adenomas (IPA) surrounding bone mass and large size, surgical resection of but not invasive to the bone, and noninvasive pituitary ade- BIPAs is a significant challenge and may increase the potential nomas (NIPA). The criteria of bone invasion included severe risk of cerebrospinal fluid leaks. So, it is important to explore destruction of the clivus or anterior skull base on CT and bone and analyze the prognosis, clinical features, and molecular destruction observed during operation or pathological reports mechanism of BIPAs to design and implement appropriate showing bone tissue infiltration (Fig. 1). This study was therapeutic interventions. In this study, we found that NGTR conducted in accordance with established ethical guidelines and invasion were independent risk factors of pituitary ade- as outlined in the Declaration of Helsinki. We obtained nomas, BIPAs had worse PFS than did NBIPAs in the NGTR written informed consent from each subject, and the Ethics and NFPA groups. Also, we identified TNFa as a key molecule Committee of Beijing Tiantan Hospital approved this study. involved in BIPAs. NR_033258, lncRNA SNHG24, miR-181c- 5p, and miR-454-3p can regulate TNFa expression. TNFa and Tissue samples and histology its related lncRNAs and miRNAs represent potential therapeu- All samples were obtained from the Beijing Tiantan Hospital tic targets for bone-invasive pituitary adenomas in the future. Neurosurgery. Fresh tumor tissue samples were immediately snap-frozen in liquid nitrogen and stored at À80C. A total of 74 pituitary adenomas samples were included in this study, of which 10 frozen tissue samples were subjected to transcriptome osteoclast differentiation and bone resorption (15, 16). In addi- microarrays (experimental group, five BIPAs and five NBIPAs; tion, lncRNAs are involved in regulating mRNA stabilization and Supplementary Table S1), 59 paraffin tissue samples were sub- transport as well as miRNA sponging (17). In this study, we found jected to IHC (confirmation group, including 27 BIPAs and 32 that NR_033258, lncRNA SNHG24, miR-181c-5p, and miR-454- NBIPAs), and five invaded bone and normal bone samples from 3p can regulate TNFa expression. BIPAs were subjected to HE staining and SEM. Materials and Methods ceRNA microarrays Patients Total RNA was extracted and purified by using the mirVana We retrospectively reviewed the patients with pituitary ade- miRNA Isolation Kit without phenol (Cat # AM1561, Ambion, noma in a single ward of Beijing Tiantan Hospital from Austin, TX, US). RNA samples from each group were then used to Figure 1. The imaging performance of three groups: A, BIPAs; B, IPAs but not invasive bone; C, NIPAs; D, Intraoperative image: bone destruction of clivus; E, HE staining of the BIPA: pituitary adenoma infiltrating bone tissue; F, electron microscope of the BIPA: diffused distribution of GH, PRL, and FSH granules. OF2 Clin Cancer Res; 2018 Clinical Cancer Research Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 6, 2018; DOI: 10.1158/1078-0432.CCR-18-0472 The Prognosis and Mechanism of BIPAs generate fluorescence-labeled cRNA targets for the SBC human ¼ CT (gene of interest) À CT (GAPDH)]. All qRT-PCR analyses ceRNA array V1.0 (4 Â 180 K). The labeled cRNA targets were then were performed in triplicate. Student t tests were applied, and a hybridized with the slides. After hybridization, the slides were P-value <0.05 was considered significant. The primer sequences scanned on the Agilent Microarray Scanner (Agilent Technolo- are presented in Supplementary Table S2. gies). The data were extracted with Feature Extraction software 10.7 (Agilent Technologies). Raw data