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1 Precise Let-7 Expression Levels Balance Organ Regeneration Against Tumor Suppression 2 3 Linwei Wu1,2,3*, Liem H

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1 Precise Let-7 Expression Levels Balance Organ Regeneration Against Tumor Suppression 2 3 Linwei Wu1,2,3*, Liem H 1 Precise let-7 expression levels balance organ regeneration against tumor suppression 2 3 Linwei Wu1,2,3*, Liem H. Nguyen1,3*, Kejin Zhou4, T. Yvanka de Soysa5, Lin Li1,3, Jason B. 4 Miller4, Jianmin Tian6, Joseph Locker6, Shuyuan Zhang1,3, Gen Shinoda5, Marc T. 5 Seligson5, Lauren R. Zeitels3,7, Asha Acharya3,7, Sam C. Wang1,3,8, Joshua T. Mendell3,7, 6 Xiaoshun He2, Jinsuke Nishino9, Sean J. Morrison9, Daniel J. Siegwart4, George Q. Daley5, 7 Ng Shyh-Chang5,10, and Hao Zhu1,3 8 9 1Children’s Research Institute, Departments of Pediatrics and Internal Medicine, Simmons 10 Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 11 75390, USA. 12 2Organ Transplant Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 13 China, 510080. 14 3Center for Regenerative Science and Medicine, University of Texas Southwestern Medical 15 Center, Dallas, Texas 75390, USA. 16 4Simmons Comprehensive Cancer Center, Department of Biochemistry, The University of Texas 17 Southwestern Medical Center, Dallas, TX 75390, USA. 18 5Stem Cell Transplantation Program, Division of Pediatric Hematology/Oncology, Boston 19 Children’s Hospital and Dana Farber Cancer Institute, Boston, Massachusetts, USA. 20 Harvard Stem Cell Institute, Boston, Massachusetts, USA. 21 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 22 Boston, Massachusetts, USA. 23 Manton Center for Orphan Disease Research, Boston, Massachusetts, USA. 24 Howard Hughes Medical Institute, Boston, Massachusetts, USA. 25 6Department of Pathology, University of Pittsburg, Pittsburg, Pennsylvania, USA. 26 7Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, 27 Texas 75390, USA; Simmons Cancer Center. 28 8Department of Surgery, The University of Texas Southwestern Medical Center, Dallas, TX 29 75390, USA. 30 9Howard Hughes Medical Institute, Children’s Research Institute, Department of Pediatrics, 31 University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 32 10Stem cell and regenerative biology, Genome Institute of Singapore, 60 Biopolis St 138672, 33 Singapore. 34 1 35 * These authors contributed equally to this work 36 37 Correspondence: 38 Hao Zhu 39 Email: [email protected] 40 Phone: (214) 648-2569 41 Fax: (214) 648-5517 42 2 43 ABSTRACT 44 The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, 45 analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs 46 tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in 47 compromised protection against cancer or tissue damage, respectively. Modest let-7 48 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive 49 oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 50 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. 51 let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR 52 pathway. We found that chronic high-dose let-7 overexpression caused liver damage and 53 degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in 54 tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, 55 and have important implications for let-7 based therapeutics. 56 3 57 INTRODUCTION 58 MicroRNAs are thought to control cellular responses to stresses such as tissue damage and 59 transformation (Leung and Sharp, 2010; Chivukula et al., 2014), but the impact of this idea is 60 unclear because microRNAs have been understudied in vivo. let-7 is one of the most ancient 61 and omnipresent microRNAs, yet relatively little is known about its functional roles in 62 mammalian development and physiology. let-7 was first identified as a gene that regulates the 63 timing of developmental milestones in a C. elegans screen (Reinhart et al., 2000). In mammals, 64 mature let-7 is undetectable in early embryos and embryonic stem cells, but becomes highly 65 expressed in most adult tissues (Schulman et al., 2005; Thomson et al., 2006). A handful of 66 previous studies have implicated let-7 in body size regulation, metabolism, stem cell self- 67 renewal, and colon carcinogenesis (Zhu et al., 2011; Frost and Olson, 2011; Nishino et al., 68 2013; Shyh-Chang et al., 2013; Madison et al., 2013), but the core functions of let-7 in 69 regeneration and disease remain incomplete. 70 71 In addition to questions about what let-7 does, it is unknown why so many let-7s are expressed 72 at such high levels. In mice and humans, the let-7 family is comprised of 10 to 12 members who 73 are thought to share a common set of mRNA targets. It has been thought that deep redundancy 74 might make it difficult to discern any phenotypes that individual let-7s might have. Essential 75 unanswered questions regarding let-7 biology include whether let-7 members are redundant, 76 have unique functions, or are regulated to maintain a specific total dose. Our previous study of 77 Lin28a, which inhibits the biogenesis of each let-7 member similarly (Heo et al., 2008; Nam et 78 al., 2011), suggests that total let-7 dose alterations, rather than regulation of specific members, 79 is important. In transgenic mice, modest increase in Lin28a and consequent 40% suppression of 80 total let-7 levels promote increased glucose uptake and an overgrowth syndrome (Zhu et al., 81 2010). 82 83 In this study we examined the consequences of let-7 dose disruption in cancer and organ 84 regeneration in genetic mouse models. While let-7s have been implicated as a tumor 85 suppressor, this has predominantly been shown in cell lines and xenograft assays (Guo et al., 86 2006; Chang et al., 2009; Iliopoulos et al., 2009; Viswanathan et al., 2009; Wang et al., 2010; 87 Lan et al., 2011), as well as using exogenous let-7 delivery to mouse cancer models (Esquela- 88 Kerscher et al., 2008; Trang et al., 2010; Trang et al., 2011). Here, we confirmed the tumor 89 suppressor activity of an endogenous transgenic let-7 in a MYC-driven hepatoblastoma model. 90 However, we found that this same level of let-7 overexpression impaired liver regeneration after 4 91 partial hepatectomy (PHx). Furthermore chronic, high-dose let-7 resulted in severe liver damage 92 and paradoxical liver cancer development. Overall, we provide in vivo evidence that let-7 93 expression levels have been developmentally constrained to balance the need for regenerative 94 proliferation against the need to antagonize malignant proliferation, findings with implications for 95 let-7 based therapies. 96 97 RESULTS 98 let-7g inhibits the development of MYC-driven hepatoblastoma 99 To study the effect of let-7 on carcinogenesis, we employed an inducible MYC-driven 100 hepatoblastoma model (Shachaf et al., 2004). In this model, most let-7s are suppressed by 101 more than 60% (Nguyen et al., 2014). However, MYC affects the expression of many other 102 microRNAs (Chang et al., 2009; Kota et al., 2009). To test if let-7 suppression is specifically 103 required for MYC’s oncogenic program, we simultaneously overexpressed let-7g and MYC 104 using a triple transgenic, liver-specific, tet-off model (Figure 1A: LAP-tTA; TRE-MYC; TRE-let- 105 7S21L transgenic mice). This transgenic form of let-7g is an engineered let-7 species called let- 106 7S21L (let-7g Stem + miR-21 Loop) (Zhu et al., 2011), in which the precursor microRNA loop 107 derives from mir-21 and cannot be bound and inhibited by Lin28b (Figure 1B), which is highly 108 expressed in MYC-driven tumors (Chang et al., 2009; Nguyen et al., 2014). 109 110 Induction of MYC with or without let-7S21L was initiated at 2 weeks of age (Figure 1C). By 90 111 days of age, large multifocal tumors had formed in 88% of the MYC alone group, whereas single 112 small tumors appeared in only 27% the MYC + let-7S21L group (Figure 1D-F) and overall 113 survival was dramatically improved (Figure 1G). The level of let-7g was increased more than 8- 114 fold in both non-tumor and tumor tissues (Figure 1H). Tumors from both groups showed similar 115 histology (Figure 1-figure supplement 1) and MYC expression (Figure 1I). Gene-expression 116 within tumors showed that previously validated let-7 targets involved in proliferation and growth 117 including Cdc25a (Johnson et al., 2007), Cdc34 (Legesse-Miller et al., 2009), E2f2 (Dong et al., 118 2010), E2f5 (Kropp et al., 2015), and Map4k4 (Tan et al., 2015) were upregulated in MYC- 119 tumors, but suppressed back down to normal levels in the context of let-7 overexpression 120 (Figure 1J-L), suggesting that the repression of these targets restrains MYC-dependent 121 tumorigenesis. These data indicated that let-7g has potent tumor suppressor activity in the 122 context of MYC-driven hepatoblastoma. 123 124 5 125 let-7g overexpression inhibits liver regeneration after partial hepatectomy 126 Since increasing let-7g was extremely effective at suppressing hepatoblastoma without 127 compromising overall health, we asked if there were consequences associated with higher let-7 128 levels. We examined let-7g overexpression in the setting of liver injuries that drive rapid 129 proliferation and growth. After PHx, let-7’s in regenerating tissues fell, but returned to normal 130 after forty hours (Figure 2A), findings consistent with previous reports (Chen et al., 2011). 131 Similarly, let-7 also declined acutely after chemical injury with the xenobiotic TCPOBOP (1,4- 132 bis-[2-(3,5-dichloropyridyloxy)] benzene) (Figure 2-figure supplement 1A). This shows that while 133 let-7 increases in a temporally defined fashion during development (Figure 2-figure supplement 134 1B), it can transiently fluctuate after environmental perturbations.
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