26 1 Endocrine-Related X Wei, S Cai et al. KAT5 in anaplastic thyroid 26:1 141–151 Cancer carcinoma RESEARCH KAT5 promotes invasion and metastasis through C-MYC stabilization in ATC Xi Wei1,*, Shang Cai2,3,*, Rebecca J Boohaker2, Joshua Fried2, Ying Li4, Linfei Hu5, Yi Pan5, Ruifen Cheng5, Sheng Zhang1, Ye Tian3, Ming Gao5 and Bo Xu2,6 1Department of Diagnostic and Therapeutic Ultrasonography, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China 2Department of Oncology, Southern Research Institute and Cancer Cell Biology Program, the University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, Alabama, USA 3Department of Radiotherapy and Oncology, the Second Affiliated Hospital ofoochow S University, Suzhou, China 4The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China 5Department of Thyroid Tumor, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, China 6Department of Molecular Radiation Oncology, Key Laboratory of Breast Cancer Prevention and Therapy, Ministry of Education, National Clinical Research Center of Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China Correspondence should be addressed to B Xu or M Gao: [email protected] or [email protected] or [email protected] *(X Wei and S Cai contributed equally to this work) Abstract Anaplastic thyroid cancer (ATC) is an aggressive cancer with poor clinical prognosis. Key Words However, mechanisms driving ATC aggressiveness is not well known. Components of f anaplastic thyroid cancer the DNA damage response (DDR) are frequently found mutated or aberrantly expressed f C-MYC in ATC. The goal of this study is to establish the functional link between histone f KAT5 acetyltransferase lysine (K) acetyltransferase 5 (KAT5, a critical DDR protein) and ATC f invasion invasiveness using clinical, in vitro and in vivo models. We analyzed the expression of f metastasis KAT5 by immunohistochemistry and assessed its relationship with metastasis and overall survival in 82 ATC patients. Using cellular models, we established functional connection of KAT5 expression and C-MYC stabilization. We then studied the impact of genetically modified KAT5 expression on ATC metastasis in nude mice. In clinical samples, there is a strong correlation of KAT5 expression with ATC metastasis (P = 0.0009) and overall survival (P = 0.0017). At the cellular level, upregulation of KAT5 significantly promotes thyroid cancer cell proliferation and invasion. We also find that KAT5 enhances the C-MYC protein level by inhibiting ubiquitin-mediated degradation. Further evidence reveals that KAT5 acetylates and stabilizes C-MYC. Finally, we prove that altered KAT5 expression influences ATC lung metastases in vivo. KAT5 promotes ATC invasion and metastases through stabilization of C-MYC, demonstrating it as a new biomarker and therapeutic target for ATC. Endocrine-Related Cancer (2019) 26, 141–151 Introduction Highly aggressive, anaplastic thyroid cancer (ATC) is capacity (Xing 2013, Molinaro et al. 2017). ATC is a rare form of thyroid cancer with median survival of typically resistant to multimodality therapies such as only ~4 months due to invasive nature and metastatic radiotherapy and chemotherapy, leaving it a continuous https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-18-0193 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 12:12:49PM via free access -18-0193 Endocrine-Related X Wei, S Cai et al. KAT5 in anaplastic thyroid 26:1 142 Cancer carcinoma challenge to successful treatment. Morphologically, ATC conclusions. In this study, we analyzed samples from is characterized by multiple atypical mitotic nuclei and 82 ATC patients in Tianjin Cancer Hospital and found osteoclast-like multinuclear giant cells. During thyroid a strong correlation of KAT5 overexpression and poor cancer evolution, the process from differentiated to clinical prognosis. KAT5 promotes ATC cell invasion and undifferentiated thyroid carcinoma undergoes molecular proliferation through acetylating C-MYC, thus inhibiting and genetic alterations (Kitahara & Sosa 2016). Multiple ubiquitination proteasome degradation and stabilizing specific genetic mutations have been identified, such as the protein. Furthermore, we demonstrate evidence that BRAF, RAS, TERT, PIK3CA, PTEN and ALK genes in ATC overexpression of KAT5 promotes metastases in an ATC (Guerra et al. 2013). Because of the rapid progression and xenograft model. high invasiveness nature of ATC, at the time of diagnosis, most patients have already contracted extra-thyroid invasion and/or lymph metastasis, and many of them Methods and materials have distant metastases (Smallridge et al. 2009). Therefore, Patient information and database analysis there is an urgent need to understand the molecular mechanism(s) driving ATC aggressiveness and to identify A total of 82 patients diagnosed with ATC between 2001 novel targets to improving the treatment outcome. and 2013 in Tianjin Medical University Cancer Hospital Genetic instability is one of the hallmarks of cancers were enrolled in the study. Evaluation of the clinical and may be associated with the underlying cause for ATC cases was approved by the Tianjin Medical University aggressiveness (Hanahan & Weinberg 2011). The DNA Institutional Cancer Hospital Review Board. The average damage response (DDR), a major mechanism for the age is 61.8 years (range from 17 to 89 years old). The mean maintenance of genome stability, contains multiple signal follow-up time is 61 months. The overall survival (OS) transduction pathways mediating cell cycle checkpoints, time ranges from 19 days to 44 months and the median DNA repair and programmed cell death (Lord & Ashworth OS is 6 months. All human tumor tissues were obtained 2012). Abnormal DDR pathways have been documented with written informed consent, prior to participation in in the majority of cancers, including ATC (Molinaro et al. the study. In the process of identifying potential DDR 2017). It is widely accepted that optimal DDR mechanisms genes in ATC that might be associated with the clinical play an essential role in prevention of tumorigenesis in aggressiveness, we analyzed the Cancer Genome Atlas the early stage of tumor development (Bartek et al. 2007b). (TCGA) database and found a cluster of genes affected in Growing evidence has also proved that hyperactivation of the DDR pathway. the DDR cascade also contributes to tumor progression and metastasis (Boohaker & Xu 2014). One critical Tissue selection and immunohistochemistry element in the DDR pathway is lysine (K) acetyltransferase 5 (KAT5), which belongs to the MYST family of histone Two experienced pathologists (P Y and Ruifen Cheng) acetyltransferase (Sun et al. 2010). Also known as Tip60, reviewed hematoxylin-eosin (HE) staining slides to KAT5 is considered a tumor suppressor gene (Gorrini decide which tissue is representative for ATC. Decided by et al. 2007), where it contributes to an important enzyme microscopic findings, the criterion was that selected tissue in repairing DNA and returning cellular function to blocks should contain more tumor tissues, less stromal normal through its regulation of the ataxia telangiectasia components, less necrosis and less hemorrhage. mutant (ATM) protein kinase (Sun et al. 2005, 2007, Sections of formalin-fixed tissues from all cases were 2009, Bhoumik et al. 2008, Kaidi & Jackson 2013). The performed using a standard immunohistochemistry role of KAT5 in cancer progression is less known. Recent protocol (Wei et al. 2014). Briefly, the 3 mm tissue sections studies have shown that KAT5 is linked to malignancy in on coated slides were heated for antigen retrieval, some cancers, such as pleural mesothelioma and prostate pretreated with a 3% solution of hydrogen peroxide cancer (Shiota et al. 2010, Cregan et al. 2016) and might for 5–10 min, rinsed and incubated with 10% bovine serve as a potential drug target in human breast cancer serum albumin (BSA) as a blocking agent. The sections and glioblastoma (Pandey et al. 2015, Sun et al. 2015, were incubated with rabbit anti-KAT5 antibody (1:100; Takino et al. 2016). Cell Signaling) for 1 h at room temperature. All steps Due to the rarity of ATC, most of the clinical studies were preceded by rinsing of sections with PBS (pH 7.6). investigating the impact of genetic alterations are limited The chromogen was 3,3-diaminobenzidine (DAB). The by sample size, making it difficult to draw clinical immunoreaction for KAT5 in the nucleus of tumor cells was https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-18-0193 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 12:12:49PM via free access Endocrine-Related X Wei, S Cai et al. KAT5 in anaplastic thyroid 26:1 143 Cancer carcinoma evaluated independently by two experienced pathologists CCA CTT TGT CAA GCT CA-3′; GAPDH reverse primer: and scored by the H-score method that combines the 5′- AGG GGT CTA CAT GGC AAC TG-3′. values of immunoreaction intensity and the percentage of tumor cell stained as described