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MAFB Promotes Cancer Stemness and Tumorigenesis in Osteosarcoma Through a Sox9-Mediated Positive Feedback Loop

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MAFB Promotes Cancer Stemness and Tumorigenesis in Osteosarcoma Through a Sox9-Mediated Positive Feedback Loop Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MAFB promotes cancer stemness and tumorigenesis in osteosarcoma through a Sox9-mediated positive feedback loop Yanyan Chen1,†, Bin Wang2, †, Mengxi Huang1, †, Tao Wang 2, †, Chao Hu 3,†, Qin Liu 2, Dong Han 4, Cheng Chen1, Junliang Zhang5, Zhiping Li4, Chao Liu 6, Wenbin Lei 7,Yue Chang1, Meijuan Wu1,Dan Xiang1, Yitian Chen1, Rui Wang1, Weiqian Huang5, Zengjie Lei1 ,* and Xiaoyuan Chu1, * 1 Department of Medical Oncology, Affiliated Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu Province, People’s Republic of China 2 Department of Gastroenterology, Daping Hospital, Third Military Medical University (Ar- my Medical University), Chongqing, People’s Republic of China 3 Department of Orthopedics, 904 Hospital of PLA, North Xingyuan Road, Beitang Dis- trict, Wuxi, Jiangsu, People’s Republic of China 4 Department of Medical Oncology, Jinling Hospital, Nanjing Clinical School of Southern Medical University, Nanjing, Jiangsu Province, People’s Republic of China 5 Department of Orthopedics, Affiliated Jinling Hospital, Medical School of Nanjing Univer- sity, Nanjing, Jiangsu Province, People’s Republic of China 6 Department of Medical Oncology, Jinling Hospital, Nanjing Clinical School of Nanjing Medical University, Nanjing, Jiangsu Province, People’s Republic of China 7Department of Orthopedics, Tianshui Cooperation of Chinese and Western Medicine Hospi- tal, Tianshui, Gansu Province, People’s Republic of China * To whom correspondence should be addressed. Xiaoyuan Chu, Tel: +86-25-80860131, Fax: +86-25-80860131, Email: [email protected]; Zengjie Lei, Email: leiz- [email protected] ; †The authors wish it to be known that, in their opinion, the first five authors should be regard- ed as joint First Authors. Running title: MAFB-Sox9 positive feedback loop promotes osteosarcoma cell stemness Keywords: osteosarcoma, transcriptional feedback loop, cell stemness, MAFB, Sox9 1 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Financial support: This work was supported by the National Natural Science Foundation of China [81572457 and 81872042 to Xiaoyuan Chu, 81702442 to Zengjie Lei, 81822032 to Bin Wang], the Natural Science Foundation of Jiangsu province [BK20170623 to Zengjie Lei], and the funding from the Army Medical University [Nos. 2019XQY19, 2018XLC2023 and 2019CXLCA001 to Bin Wang]. Conflicts of interest: The authors declare no conflicts of interest. Word count: 4798 6 figures, 5 Supplementary Figures, 3 Supplementary Tables. 2 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Significance Transcription factors MAFB and Sox9 form a positive feedback loop to maintain cell stem- ness and tumor growth in vitro and in vivo, revealing a potential target pathway for therapeu- tic intervention in osteosarcoma.. 3 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Despite the fact that osteosarcoma is one of the most common primary bone malignancies with poor prognosis, the mechanism behind the pathogenesis of osteosarcoma is only partial- ly known. Here we characterized differentially expressed genes (DEG) by extensive analysis of several publicly available gene expression profile datasets and identified MAFB as a key transcriptional regulator in osteosarcoma progression. MAFB was highly expressed in tumor tissues and required for proliferation and tumorigenicity of osteosarcoma cells. MAFB ex- pression was elevated in osteosarcoma stem cells to maintain their self-renewal potential in vitro and in vivo through upregulation of stem cell regulator Sox9 at the transcriptional level. Sox9 in turn activated MAFB expression via direct recognition of its sequence binding en- richment (SBE) motif on the MAFB locus, thereby forming a positive feedback regulatory loop. Sox9-mediated feedback activation of MAFB was pivotal to tumorsphere-forming and tumor-initiating capacities of osteosarcoma stem cells. Moreover, expression of MAFB and Sox9 was highly correlated in osteosarcoma and associated with disease progression. Com- bined detection of both MAFB and Sox9 represented a promising prognostic biomarker that stratified a subset of osteosarcoma patients with shortest overall survival. Taken together, these findings reveal a MAFB-Sox9 reciprocal regulatory axis driving cancer stemness and malignancy in osteosarcoma and identify novel molecular targets that might be therapeutical- ly applicable in clinical settings. 4 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Osteosarcoma is one of the most common primary malignant bone tumors with two incidence peaks, one in adolescence and another in the elderly population, especially those above 75 years of age (1). Chemotherapy combined with surgery has greatly improved clinical out- comes of osteosarcoma patients, with the 5-year survival rate up to 60-70%, while ~20% of osteosarcoma patients with metastasis continue to have poor prognosis (2). Increasing clinical and experimental evidence indicates that osteosarcoma stem cells, which derive from mesenchymal stem cells, may be the cellular origin of osteosarcomas (3). Cancer stem cells (CSCs) share many similar properties with normal stem cells and are pri- marily responsible for tumorigenesis in many cancers (4, 5). For example, a subpopulation of self-renewing osteosarcoma cells, namely CSCs, are endowed with intrinsic capacities for tumor initiation and drug resistance (6, 7). These CSCs are regulated by several key tran- scription factors and signal pathways, such as Oct3/4, Sox2, Nanog, and Notch (8). Com- pared to differentiated cancer cells, CSCs are generally more malignant and are critical de- terminants of the response to chemotherapy and radiotherapy, and therefore the eradication of osteosarcoma stem cells may be an effective treatment strategy (9, 10). V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a member of the MAF transcription factor family, containing basic leucine zipper domains which bind to specific DNA elements (11). The seven MAF members are separated into two classes (small and large MAFs). Small MAF proteins (MAFF, MAFG, and MAFK) have been shown to regulate antioxidant responses (12) whereas large MAFs (MAFA, c-MAF, and MAFB) each contain a similar transactivation domain and are strongly oncogenic (13, 14). In the hematopoietic system, MAFB induces myelomonocytic differentiation in immortalized myoblasts and macrophage differentiation and maturation in mice (15). In podocyte differen- tiation and the maintenance of progression, aberrant podocyte foot process formation is ob- 5 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. served in a MAFB-mutant zebrafish embryo model (15). There is also evidence that MAFB regulates osteoclast genesis and epidermal keratinocyte differentiation (16, 17). Up- regulation of MAFB increases the risk of various human pathologies, such as diabetes and atherosclerotic disorders (18, 19). In addition, MAFB has been shown to promote tumorigen- esis, especially in the transformation of pancreatic β cells (14, 20). MAFB chromosomal translocations occur more frequently in human myeloma cells, and high expression of MAFB is observed in acute leukemia blast, hepatocellular carcinoma, and colorectal carcinoma pa- tients (15, 18). Finally, MAFB has been shown to promote nasopharyngeal carcinoma cell proliferation and migration (21). Here, through high throughput bioinformatic analysis of publicly available transcriptome datasets we identified MAFB as a novel regulator of osteosarcoma tumorigenesis. We demonstrate that MAFB promotes tumorigenesis and self-renewal of osteosarcoma stem cells via a Sox9-mediated feedback activation loop, which could be exploited to eliminate the cul- prit cells in osteosarcoma. 6 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 31, 2020; DOI: 10.1158/0008-5472.CAN-19-1764 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Collection and processing of microarray
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