Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway ... - Cell Press

Article

Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway that Is Cell-Density Dependent and Hippo Independent Graphical Abstract

Authors Hiroki Hikasa, Yoshitaka Sekido, Akira Suzuki

Correspondence [email protected]

In Brief Hikasa et al. reveal an essential growthinhibitory mechanism, by which Merlin/ NF2 suppresses Lin28B function and promotes let-7 biogenesis in a YAP1/ TAZ-independent manner. Contact inhibition triggers Merlin/NF2 dephosphorylation, which inhibits nucleolar/nuclear localization of Lin28B and induces pri-let7 maturation, leading to cell growth inhibition. They suggest that Merlin/NF2 thereby drives a celldensity-dependent tumor-suppressive pathway.

Highlights d

Lin28B, which inhibits let-7 microRNA, is a key downstream target of Merlin/NF2

d

At low cell density, phosphorylated Merlin/NF2 does not bind to Lin28B

d

Dephosphorylated Merlin/NF2 sequesters Lin28B, inhibiting growth

d

Merlin/NF2 drives cell-density-dependent and Hippoindependent tumor suppression

Hikasa et al., 2016, Cell Reports 14, 2950–2961 March 29, 2016 ª2016 The Authors http://dx.doi.org/10.1016/j.celrep.2016.02.075

Cell Reports

Article Merlin/NF2-Lin28B-let-7 Is a Tumor-Suppressive Pathway that Is Cell-Density Dependent and Hippo Independent Hiroki Hikasa,1 Yoshitaka Sekido,2 and Akira Suzuki1,* 1Division of Cancer Genetics, Medical Institute of Bioregulation, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan 2Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.02.075 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

SUMMARY

Contact inhibition of proliferation is critical for tissue organization, and its dysregulation contributes to tumorigenesis. Merlin/NF2 is a tumor suppressor that governs contact inhibition. Although Merlin/ NF2 inhibits YAP1 and TAZ, which are paralogous Hippo pathway transcriptional co-activators and oncoproteins, it is not fully understood how Merlin/NF2mediated signal transduction triggered by cell-cell contact exerts tumor suppression. Here, we identify Lin28B, an inhibitor of let-7 microRNAs (miRNAs), as an important downstream target of Merlin/NF2. Functional studies revealed that, at low cell density, Merlin/NF2 is phosphorylated and does not bind to Lin28B, allowing Lin28B to enter the nucleus, bind to pri-let-7 miRNAs, and inhibit their maturation in a YAP1/TAZ-independent manner. This inhibition of pri-let-7 maturation then promotes cell growth. However, cell-cell contact triggers Merlin/NF2 dephosphorylation, which sequesters Lin28B in the cytoplasm and permits pri-let-7 maturation. Our results reveal that Merlin/NF2-mediated signaling drives a tumor-suppressive pathway that is cell-density dependent and Hippo independent. INTRODUCTION Intercellular contact leads to inhibition of cell proliferation, a process called contact inhibition. Contact inhibition is essential for the maintenance of tissue homeostasis, and its disruption is characteristic of cancer (Hanahan and Weinberg, 2011). Merlin, also known as neurofibromatosis type 2 (NF2), is a major effector of contact inhibition and also a tumor suppressor gene (Curto et al., 2007; Morrison et al., 2001; Okada et al., 2005; Yi et al., 2011). The presence of inactivated Merlin/NF2 in a variety of cancers in both mice and humans is evidence of Merlin/NF2’s potent tumor-suppressive activity (Li et al., 2012; Sekido, 2013).

Merlin/NF2 is a FERM-domain-containing protein and serves as a linker between transmembrane proteins and a cell’s actin cytoskeleton. Merlin/NF2 inhibits mitogenic signal transduction, and several lines of evidence suggest that this growthinhibitory role is associated with receptor-dependent pathways such as EGFR-RAS signaling (Benhamouche et al., 2010; Curto et al., 2007), Rac-PAK signaling (Kissil et al., 2003; Okada et al., 2005; Xiao et al., 2002), and HA-CD44 signaling (Bai et al., 2007; Morrison et al., 2001). Furthermore, there is genetic evidence in mice and flies that Merlin/NF2 activates the tumor-suppressive Hippo pathway that is important for contact inhibition. This Hippo pathway activation leads to inhibition of the paralogous transcriptional coactivators YAP1 and TAZ, which are key regulators of cell proliferation (Hong and Guan, 2012; Pellock et al., 2007; Yu et al., 2010; Zhang et al., 2010). In addition, recent studies have demonstrated that nuclear Merlin/NF2 inhibits the CRLDCAF1-mediated proteolysis pathway critical for cell growth (Li et al., 2010, 2014). However, although it is clear that Merlin/NF2 functionally interacts with a variety of signal-transduction pathways controlling cell proliferation and survival, it remains to be elucidated which pathway contributes to Merlin/NF2’s tumorsuppressive activity during contact-mediated inhibition of cell proliferation. There is emerging evidence that the RNA-binding protein Lin28 is involved in cell growth and reprogramming, as well as in tissue homeostasis and cancer development (Rehfeld et al., 2015; Thornton and Gregory, 2012; Zhou et al., 2013). When Lin28 is overexpressed in mice, it enhances the metabolism of glucose and other bioenergetic molecules, leading to delayed onset of puberty, resistance to the development of obesity and diabetes, and enhanced tissue repair (Shyh-Chang et al., 2013; Zhu et al., 2010, 2011). In addition, mice with tissue-specific overexpression of Lin28 in neural crest, liver or small intestine develop tumors (Madison et al., 2013; Molenaar et al., 2012; Nguyen et al., 2014). Mechanistically, Lin28 regulates the translation of various mRNAs (Cho et al., 2012; Graf et al., 2013; Mayr and Heinemann, 2013; Shyh-Chang et al., 2013; Wilbert et al., 2012) and specifically suppresses the biogenesis of the let-7 microRNA (miRNA) family (Roush and Slack, 2008; Viswanathan and Daley, 2010). Let-7 miRNAs act as tumor suppressors by silencing the expression of critical oncogenes such as Myc,

2950 Cell Reports 14, 2950–2961, March 29, 2016 ª2016 The Authors

A

B

C

D

Figure 1. Merlin/NF2’s FERM and a-Helix Domains Mediate Interaction with Lin28B’s Cold Shock Domain (A) Immunoblot (IB) to detect the indicated proteins in lysates of HEK293T cells that expressed myc-tagged Lin28B plus flag-tagged Merlin/NF2 (flagMer) or flagtagged MST1 and were subjected to immunoprecipitation (IP) with anti-flag Ab (aflag). GFP, transfection control. For all IB, results are representative of three experiments. (B) IB to detect the indicated endogenous proteins in lysates of H1299 cells subjected to IP with anti-Lin28B Ab or control IgG. (C) IB of H1299 cells that were infected with lentivirus expressing the indicated flagMer constructs and immunoprecipitated with anti-flag Ab to detect the binding of endogenous Lin28B to flagMer constructs. Mer WT, wild-type Merlin/NF2 protein showing the FERM (F for 4.1 protein, E for ezrin, R for radixin and M for moesin), a-helix and C-terminal domains. Mer DF, Merlin/NF2 protein missing 91 amino acids of the FERM domain; Mer DH, Merlin/NF2 protein missing the entire a-helix domain; Mer DC, Merlin/NF2 protein missing the entire C-terminal domain. (D) IB to detect the indicated proteins in HEK293T cells that were co-transfected with the indicated mycLin28B constructs plus GFP or flagMer, and immunoprecipitated with anti-flag Ab. Lin28B WT, wild-type Lin28B protein showing the CSD (cold shock domain), L (linker domain), Zinc finger (Zinc F), and C-terminal domains. Lin28B DC, Lin28B protein missing the entire CSD domain; Lin28B DL, Lin28B protein missing the entire linker domain; Lin28B DZ, Lin28B protein missing the entire zinc-finger domain plus 77 amino acids of the C-terminal domain.

HMGA2, IMP1, and RAS (Johnson et al., 2005; Kumar et al., 2007; Lee and Dutta, 2007; Nishino et al., 2008). Lin28 inhibits the synthesis of the mature form of let-7 (mlet-7) at two distinct subcellular levels. In the nucleus, Lin28 associates with primary let-7 (pri-let-7) to block Drosha-mediated processing of this molecule into precursor let-7 (pre-let-7) (Van Wynsberghe et al., 2011; Viswanathan et al., 2008). In the cytoplasm, Lin28 binds to pre-let-7, inducing its TUT4/7-mediated oligouridylation and rapid decay (Hagan et al., 2009; Heo et al., 2008, 2009; Lehrbach et al., 2009). There are two isoforms of mammalian Lin28: the closely related paralogs Lin28A and Lin28B. Several types of primary human tumors express either Lin28A or Lin28B, and both isoforms function as oncoproteins when expressed ectopically. Lin28A is thought to act on pri-let-7 miRNAs in the nucleus and on pre-let-7 mRNAs in the cytoplasm (Kim et al., 2014), whereas Lin28B preferentially acts on nuclear pri-let-7 miRNAs (Piskounova et al., 2011). However, the pathways regulating Lin28A/B localization and activity in response to intercellular signaling are still unknown. In this report, we demonstrate that (1) Lin28B interacts with Merlin/NF2, (2) Merlin/NF2 is a key regulator of Lin28B localization and mlet-7 biogenesis in response to cell-cell contact, and (3) Lin28-let-7 interaction makes a major contribution to Merlin/ NF2’s growth inhibitory activity. Our results show that the Merlin/NF2-Lin28-let-7 axis is a cell-density-dependent tumorsuppressive pathway.

RESULTS Merlin/NF2 Associates with Lin28B To identify signaling mediators acting downstream of Merlin/ NF2, we performed a mass spectrometry analysis of HEK293T cells (human embryonic kidney cell line) engineered to overexpress exogenous Merlin/NF2 tagged with flag epitope. In addition to previously known partners such as VprBP, DDB1, and HECT E3 ubiquitin ligase, Lin28B was isolated as a new Merlin/ NF2-interacting protein (Table S1). This association between Lin28B and Merlin/NF2 was confirmed by immunoprecipitation analyses (Figure 1A) and demonstrated for the endogenous proteins in H1299 cells (human lung cancer cell line) (Figure 1B). We then made several deletion constructs of flag-tagged Merlin/NF2 to determine which region of Merlin/NF2 was responsible for Lin28B binding. We found that this association requires parts of Merlin/NF20 s FERM domain and the adjacent a-helical domain (Figure 1C), both of which are implicated in Merlin/NF20 s proteinprotein interactions and structural conformation changes. Conversely, to identify the region of Lin28B that associates with Merlin/NF2, we examined the binding of several truncated constructs of myc-tagged Lin28B lacking its cold shock domain (mycLin28BDC), the linker domain (mycLin28BDL), or the CCHC zinc-finger domain (mycLin28BDZ) (Figure 1D). Flag-tagged Merlin/NF2 failed to bind to mycLin28BDC (Figure 1D), the construct lacking the cold shock domain essential for the physical interaction of Lin28B with the pre-let-7 family (Nam et al.,

Cell Reports 14, 2950–2961, March 29, 2016 ª2016 The Authors 2951

A

D

B

C

E

F

Figure 2. Merlin/NF2 Promotes let-7 Maturation and Suppresses Lin28B Protein Levels (A) IB to detect the indicated proteins in H1299 cells plated at high density (HD) and infected with lentivirus expressing the indicated shRNAs. Analysis was performed at 3 days post-lentivirus infection. shCont, scrambled control shRNA. (B) qPCR analysis of the indicated mature miRNAs in the H1299 cells in (A). Data were normalized to U6 snRNA and expressed relative to the level of control shRNA (shCont) set to 1. Results are the mean ± SD (n = 3). (C) IB to detect the indicated proteins in H290 cells that were plated at HD and infected with lentiviruses expressing empty mCherry vector (control), flagMerSA or flagMerSD. Analysis was performed at 3 days post-lentivirus infection. (D) qPCR analysis of the indicated mature miRNAs in the H290 cells in (C). Data were normalized as in (B) and expressed relative to the mCherry value set to 1. Results are the mean ± SD (n = 3). (E) IB to detect the indicated proteins in H1299 cells and infected with lentivirus expressing the indicated shRNAs. shY/T, shYAP/TAZ. Analysis was performed at 3 days post-lentivirus infection. (F) qPCR analysis of the indicated mature miRNAs in the H1299 cells in (E). Data were normalized and analyzed as in (B). Results are the mean ± SD (n = 3). *p < 0.05 (compared to shNF2 alone). For all figures, *p < 0.05 by Student’s t test.

2011; Piskounova et al., 2008). These findings raised the possibility that Merlin/NF2 might regulate mlet-7 biogenesis via a domain-specific interaction that inhibits Lin28B function. Merlin/NF2 Regulates mlet-7 Biogenesis via Lin28B To assess the involvement of Merlin/NF2 in regulating Lin28B and let-7, we first examined the effect of Merlin/NF2 depletion on protein levels of Lin28B and mlet-7 in H1299, Huh7 (hepatocarcinoma cell line) and Caco2 (colon cancer cell line) cells, all of which express Lin28B. Knockdown of Merlin/NF2 with short hairpin RNAs (shRNAs) resulted in a slight increase in Lin28B protein (Figures 2A, S1A, and S1C) and significant decreases in mlet-7a, 7c, and 7g miRNAs, but it had no effect on the

Lin28-independent miRNA miR-16 (Figures 2B, S1B, and S1D). To confirm these effects of Merlin/NF2 on mlet-7 miRNAs and Lin28B, we employed H290 cells, which are a malignant mesothelioma cell line with a homozygous deletion of the endogenous Merlin/NF2 gene. Using lentivirus-mediated gene expression, we engineered H290 cells to express one of two altered forms of Merlin/NF2. Constitutive expression of a growth-inhibitory (activated) form of Merlin/NF2 (termed flagMerSA), in which there is an alanine substitution at the S518 phosphorylation site, led to a striking increase in mlet-7 miRNAs and a decrease in Lin28B. Conversely, expression of a phosphomimetic (inhibited) form of Merlin/NF2 (flagMerSD) had no effect on mlet-7 miRNAs and Lin28B (Figures 2C and 2D). These data support


A

B

D

Figure 3. Cell-Cell Contact Dephosphorylates Merlin/NF2 and Promotes Physical Interaction between Merlin/NF2 and Lin28B

C

(A) IB to detect the indicated proteins in H1299 cells or H290 cells that were cultured at HD (H) or low density (LD; L) for 3 days. (B) qPCR analysis of the indicated mature miRNAs in the cells in (A). Data were normalized as in Figure 2B and are expressed relative to the LD value set to 1. Results are the mean ± SD (n = 3). *p < 0.05 (C) qPCR analysis of expression of pri-let7 miRNAs in H1299 cells and H290 cells that were cultured at LD or HD. Data were normalized to actin mRNA and are expressed relative to the LD value set to 1. Results are the mean ± SD (n = 3). (D) IB to detect the indicated proteins in H1299 cells that expressed the indicated flagMer constructs, were cultured at HD, and were immunoprecipitated with anti-flag Ab. (E) IB to detect the indicated proteins in H1299 cells that expressed the empty vector, flagMerWT or flagMerSA, were cultured at LD or HD as in (A), and were immunoprecipitated with anti-flag Ab.

E

our hypothesis that Merlin/NF2 promotes the maturation of let-7 miRNAs while decreasing Lin28 protein. Because the Hippo pathway effector YAP1 globally suppresses miRNA biogenesis in human keratinocyte HaCaT cells (Mori et al., 2014), we investigated if YAP1/TAZ were involved in Merlin/NF2-mediated regulation of mlet-7 miRNAs and Lin28B in H1299 cells. The reduction in mlet-7 miRNAs and elevation of Lin28B protein induced by Merlin/NF2 knockdown were not significantly affected by shRNA-mediated suppression of YAP1/TAZ, whereas changes to mlet-7 miRNA levels were clearly reversed by Lin28B knockdown (Figures 2E and 2F). These results show that the effects of Merlin/NF2 on mlet-7 biogenesis depend on Lin28B, but not on YAP1/TAZ. Cell-Cell Contact Dephosphorylates Merlin/NF2 at S518 and Promotes Its Association with Lin28B Since growth-inhibitory signaling triggered by cell-cell contact is known to activate Merlin/NF2 through dephosphorylation at S518 (Morrison et al., 2001), we examined whether cell-cell contact affected levels of let-7 miRNAs and Lin28B in H1299 and H290 cells. We observed that Merlin/NF2 S518 phosphorylation was inhibited in H1299 cells plated at high cell density (Figure 3A) and that H1299 cells (but not H290 cells) plated at high density showed elevated mlet-7 miRNAs (Figure 3B) and decreased Lin28B protein (Figures 3A and 3E). However, expression of pri-let-7 miRNAs showed only subtle change in either H1299 or

H290 cells under these culture conditions (Figure 3C). This finding in H1299 cells stands in contrast to a previous report (Mori et al., 2014) and may be due to differing sizes of the pri-let-7 pools in the cell lines tested. To investigate how Merlin/NF2 regulates Lin28B during contact inhibition, we determined if cell-density-dependent dephosphorylation of Merlin/NF2 at S518 altered its affinity for Lin28B. Immunoprecipitation analysis demonstrated that flagMerSA protein associated with Lin28B more effectively than did either wild-type (WT) Merlin/NF2 (flagMerWT) or flagMerSD (Figure 3D). Furthermore, flagMerWT displayed a stronger affinity for Lin28B when it was less highly phosphorylated at S518 (that is, at high cell density) than when it was phosphorylated at S518 (at low cell density) (Figure 3E). These findings suggest that cell-cell contact reduces the phosphorylation of Merlin/NF2 in a way that strengthens its association with Lin28B and inhibits Lin28B functions. Cell-Cell Contact Regulates Subcellular Localization of Lin28B via Merlin/NF2 The dynamic cell-density-dependent interaction we observed between Merlin/NF2 and Lin28B prompted us to determine whether Lin28B’s subcellular localization was altered in response to cell-cell contact. To this end, we used immunofluorescent staining to analyze the subcellular localization of endogenous Lin28B in H1299, Huh7, and Caco2 cells cultured at low or high cell density. Consistent with previous reports (Piskounova et al., 2011), in H1299 cells at low density, Lin28B was localized in the nucleoli (identified by the nuclear/nucleolar marker B23) as well as in the cytoplasm (Figures 4A and S2A). At high cell density, the nucleolar localization of Lin28B was abolished, but its presence in the cytoplasm was maintained (Figure 4A). Similar


A

D

B

C

E

F

G

Figure 4. Cell-Cell Contact and Merlin/NF2 Controls the Nucleolar Localization of Lin28B (A) Immunocytochemistry to detect endogenous Lin28B protein in the nucleoli of H1299 cells cultured at LD or HD. DAPI, nuclear stain. For all immunocytochemistry analyses, results are representative of three independent experiments. Scale bar represents 20 mm. (B) Immunocytochemistry to detect endogenous Lin28B protein in the nucleoli of H290 cells cultured at LD or HD for 3 days. Scale bar represents 20 mm. (C) Immunocytochemistry to detect endogenous Merlin/NF2 protein in H1299 cells cultured at LD or HD as in Figure 3A. At LD, Merlin/NF2 appears in filopodialike structures and throughout the nucleus and cytoplasm. At HD, Merlin/NF2 localizes at the plasma membrane in the intercellular contact region. Scale bar represents 20 mm. (D) IB to detect the indicated proteins in the cytoplasmic (C), membrane (M), and nuclear (N) fractions of H1299 cells that were cultured at LD or HD. Lamin, nuclear marker; a-tubulin, cytoplasmic marker; EGFR, plasma membrane marker. (E) Immunocytochemistry to detect endogenous Lin28B protein in H1299 cells infected with lentivirus expressing shCont or shMerlin/NF2 (shNF2) and cultured at HD for 3 days. Scale bar represents 20 mm. (F) Immunocytochemistry to detect endogenous Lin28B protein in H290 cells that expressed Dox-inducible flagMerSA (H290MerSA) and were cultured at HD for 3 days. Cells were left untreated (Dox-) or were treated with Dox for 3 days prior to immunocytochemistry (Dox+). Scale bar represents 20 mm. (G) Quantitation of BrdU incorporation in H290MerSA cells that were left untreated (Dox) or were treated with Dox for 3 days (Dox+) prior to 2 hr incubation with BrdU. Cells were immunostained with anti-BrdU and anti-Lin28B Abs, and the number of BrdU+ cells was counted. Results are the mean ± SD (n = 3). *p < 0.05. See also Figure S3B.

results were obtained using Huh7 and Caco2 cells (Figures S2B and S2C). In contrast, when we cultured Merlin/NF2-deficient H290 cells at high density, Lin28B remained in the nucleoli (Figure 4B). These results imply that Merlin/NF2 is not required for the entrance of Lin28B into the nucleolus but is necessary for its exit from this organelle. When we examined the localization of endogenous Merlin/NF2 in H1299 cells, we found that Merlin/NF2 formed filopodia-like structures and was broadly detectable in the cytoplasm of cells cultured at low density (Figure 4C, top). In contrast, in H1299 cells cultured at high density, Merlin/NF2 appeared in the plasma membrane in the region of intercellular contact (Figure 4C, bottom). To confirm these changes to Merlin/NF2 localization during contact inhibition, we prepared cytoplasmic (a-tubulin+), nuclear (lamin+), and plasma membrane (EGFR+) fractions of H1299 cell

lysates. At high cell density, both Lin28B and Merlin/NF2 were decreased in the nuclear fraction but maintained at relatively high levels in the cytoplasmic and plasma membrane fractions (Figure 4D). In contrast, these proteins were detectable in all fractions of H1299 cells at low cell density. Of note, Merlin/NF2 phosphorylated at S518 showed WT subcellular distribution (Figure 4D), implying that this phosphorylation site does not substantially regulate Merlin/NF2’s subcellular localization. Consistent with this hypothesis, both flagMerSA and flagMerSD behaved similarly in fractionated lysates of H290 cells plated at low or high density (Figure S3A). We next examined the effects of Merlin/NF2 knockdown or overexpression on subcellular Lin28B localization. The nucleolar localization of Lin28B that disappeared in high density H1299 cells was recovered in Merlin/NF2-depleted H1299 cells


A

Figure 5. Cell-Cell Contact Inhibits the Nuclear Function of Lin28B

B

(A) Immunocytochemistry to detect flagLin28BWT or flagLin28BDC in HeLa cells that were infected with lentivirus expressing the indicated proteins, co-cultured at HD or LD for 3 days with uninfected HeLa cells, and immunostained with anti-Lin28B Ab. Scale bar represents 20 mm. (B) Top: qPCR analysis of the indicated mature miRNAs in the HeLa cells in (A). Data were normalized to U6 snRNA and expressed relative to the level of empty vector (mCh) set to 1. Results are the mean ± SD (n = 3). *p < 0.05 (compared to WT at LD). Bottom: IB to detect the indicated proteins in the HeLa cells in the top panel.

(Figure 4E). In addition, nucleolar localization of Lin28B was attenuated in H290 cells expressing flagMerSA in a doxycycline (Dox)-dependent manner (H290MerSA cells) (Figure 4F), leading to a reduction in bromodeoxyuridine (BrdU)+ cells (Figures 4G and S3B). Supporting this, subcellular fractionation also exhibited that flagMerSA not only decreases the total level of Lin28B proteins but also promotes translocation of Lin28B out of nucleus, whereas flagMerSD did not alter subcellular proportion of Lin28B localization compared to negative control mCherry (Figure S3A). These data suggest that the regulation of Merlin/NF2 mediated by cell-cell contact is actively involved in translocating Lin28B out of the nucleolus. Interaction between Merlin and Lin28B Promotes Lin28B Translocation out of the Nucleus/Nucleolus, Inhibits Lin28B/pri-let-7 Interaction, and Promotes mlet-7 Biogenesis We next compared responses to cell-cell contact between WT Lin28B (flagLin28BWT) and flagLin28BDC (does not bind Merlin/NF2; Figure 1D) in HeLa cells, which lack Lin28A/B expression. Consistent with the subcellular localization of endogenous Lin28B observed in H1299, Huh7, and Caco2 cells (Figures 4A and S2A–S2C), the nucleolar localization of flagLin28BWT in low-density HeLa cells disappeared at high density, whereas its cytoplasmic localization was still detectable (Figure 5A). Moreover, the suppression of mlet-7 miRNAs by flagLin28BWT in low-density HeLa cells was impaired by cell-cell contact, with no significant change in flagLin28BWT protein (Figure 5B). In contrast, flagLin28BDC was exclusively localized in the nucleus/nucleoli regardless of cell density (Figure 5A), suggesting that Merlin/NF2 cannot move flagLin28BDC out of the nucleus even when activated. However, despite lacking the cold shock domain, flagLin28BDC was still able to suppress mlet7 miRNAs (Figure 5B), implying that the CCHC zinc finger domain can facilitate pri-let-7 binding in the nucleus. Importantly, the activity of flagLin28BDC, unlike flagLin28BWT, was not significantly affected by cell density (Figure 5B), probably

because flagLin28BDC was immune to Merlin/NF2-mediated inhibition. Finally, unlike in H1299 cells, flagMerSA did not alter mlet7 expression in HeLa cells (Figures S4A and S4B). These data further indicate that Merlin/NF2 regulates let-7 biogenesis in a Lin28Bdependent manner. Thus, Merlin/NF2’s inhibition of Lin28B functions is essential for the promotion of pri-let-7 processing that occurs at high cell density. We then investigated whether contact inhibition and/or the presence of Merlin/NF2 altered the affinity of endogenous Lin28B for the pri-let7 miRNAs that predominates in the nucleus. We devised an RNA immunoprecipitation (RIP) assay in which amounts of endogenous Lin28B protein in cell extracts were measured and equalized by immunoblotting, followed by immunoprecipitation of endogenous Lin28B protein with anti-Lin28B antibody (Ab) and analysis of associated pri-let-7 miRNAs (Figures S5A–S5C). Application of this assay to H1299 cells plated at high or low density showed that, consistent with our findings on the subcellular localization of Lin28B, the binding of Lin28B to pri-let-7 miRNAs was dramatically decreased in cells at high density compared to those at low density (Figure 6A). In addition, Merlin/NF2 depletion enhanced Lin28B binding to pri-let-7 miRNAs (Figure 6B), and flagMerSA more effectively blocked the formation of the Lin28B/pri-let-7 miRNAs complex than did flagMerWT (Figure 6C). These data collectively indicate that activation of Merlin/NF2 by cell-cell contact causes it to alter the subcellular localization of Lin28B and thereby regulate the binding of Lin28B to the pri-let-7 family. Growth-Inhibitory Activity of Merlin/NF2 Depends on Its Effects on Lin28B and mlet7 Biogenesis Given our finding that Merlin/NF2 promotes let-7 maturation by sequestering Lin28B, we investigated whether Merlin/NF’s involvement in mlet-7 biogenesis and Lin28B localization contributed to Merlin/NF2-mediated tumor suppression. We first examined the roles of Merlin/NF2 and Lin28B in the self-renewal of tumor cells as assayed by sphere formation in H1299, Huh7, and Caco2 cell cultures. Primary and/or secondary sphere formation in such cultures was improved by Merlin/NF2 knockdown but markedly inhibited by additional knockdown of Lin28B (Figures 7A and S6A–S6C) or YAP1/TAZ (Figure S6D). These data


A

B

C

Figure 6. Non-phosphorylated Merlin/NF2 Sequesters Lin28B and Prevents Its Interaction with Pri-let7 (A) Immunoprecipitation (RIP-IP, top) and qPCR (RIP-qPCR, bottom) for RIP assay examining the effect of contact inhibition on the physical interaction between Lin28B and the indicated pri-let-7 miRNAs in H1299 cells cultured at LD or HD. The amount of cell lysate used for each RIP assay was estimated according to the Lin28B level in the corresponding IB shown in Figure S5A. Binding of pri-let-7 miRNAs to Lin28B was normalized to pri-let-7 miRNA expression in the input cell lysate. Data are the mean ± SD (n = 3). (B) RIP-IP (top) and RIP-qPCR (bottom) assays examining the physical interaction between Lin28B and pri-let-7 miRNAs in H1299 cells infected with lentivirus expressing the indicated shRNAs and cultured at HD. See also Figure S5B. Data are the mean ± SD (n = 3). (C) RIP-IP (top) and RIP-qPCR (bottom) assays examining the physical interaction between Lin28B and pri-let-7 miRNAs in H1299 cells infected with lentivirus expressing empty vector or the MerWT or MerSA constructs. See also Figure S5C. Data are the mean ± SD (n = 3). For all figures, *p < 0.05.

suggest that Merlin/NF2 normally suppresses the self-renewal of tumor cells and that this self-renewal requires not only YAP1/ TAZ but also Lin28B. Next, we evaluated if Lin28B overexpression via lentiviral infection could prevent Merlin/NF2-mediated inhibition of the proliferation of H290MerSA cells. Whereas Dox-induced flagMerSA expression alone significantly reduced H290MerSA proliferation, co-expression of flagMerSA and Lin28B substantially restored cell growth (Figure 7B). These results indicate that Lin28B alone can partially compensate for loss of Merlin/NF2 function and that YAP1/TAZ may be required for complete rescue of cell growth suppressed by Merlin/NF2. Because previous reports have indicated that Lin28 targets not only the let-7 miRNA family but also a variety of mRNAs known to regulate biological events such as neurogenesis and tissue repair (Rehfeld

et al., 2015; Shyh-Chang et al., 2013), we also tested whether knockdown of let-7 miRNAs with locked nucleic acid (LNA) oligonucleotides could alter Merlin/NF2-mediated growth suppression. LNAs directed against let-7 sequences efficiently blocked endogenous expression of mlet-7 and stabilized several of its known targets, including Lin28B, IMP1, and cMyc (Figures S7A and S7B). Further supporting the biological significance of this Merlin/NF2-Lin28B-let-7 axis, LNAs restored the growth of cells expressing flagMerSA (Figure 7C). Because we confirmed that let-7g overexpression inhibits cell growth as well as the expression of mlet-7 target proteins in H1299 and H290 cells (Figures S7C and S7D), we examined the expression of these molecules in our Merlin/NF2-mediated growth inhibition assay. Recovery of Merlin/NF2 function in Dox-treated H290MerSA cells clearly decreased Lin28B, IMP1, and cMyc protein levels (Figure 7D),


A

D

B

C

E

F

Figure 7. Merlin/NF2 Inhibits Cell Proliferation in a Lin28B/let-7-Dependent Manner (A) (Left) Microscopic views of primary spheres formed in culture by H1299 cells that were infected with lentivirus expressing the indicated shRNAs for 2 days and then dissociated into single cells. The number of spheres >200 mm diameter formed in culture was counted 8 days after dissociation. (Right) Quantitation of the spheres formed in the left panel. Data are the mean ± SD (n = 3) per 1,000 cells. Scale bar represents 500 mm. (B) Quantitation of numbers of viable H290MerSA cells that were infected with empty vector or lentivirus expressing Lin28B, cultured with/without Dox for the indicated days, and stained with trypan blue on days 0, 2, 4 and 6. Data are the mean ± SD (n = 3). (C) Quantitation of numbers of viable H290MerSA cells that were transfected with control scrambled oligos (sc) or let-7 LNAs, cultured with/without Dox for the indicated days, and stained with trypan blue on days 0, 2, 4 and 6. Data are the mean ± SD (n = 3). (D) IB to detect the indicated proteins in the H290MerSA cells in (B) on day 6. (E) IB to detect the indicated proteins in the H290MerSA cells in (C) on day 6. (F) Proposed model of how Merlin/NF2 might regulate Lin28B and mlet7 biogenesis during contact inhibition. Please see main text for description. For all figures, *p < 0.05.

and these reductions did not occur in cells expressing exogenous Lin28B or let-7 LNAs (Figure 7E). These functional data strongly suggest that Merlin/NF2 exerts its tumor-suppressive activity by sequestering Lin28B and thus blocking its functions in the nucleus/nucleoli. Such a decrease in Lin28B activity would lead to an increase in mlet-7 and a subsequent reduction in let-7 target proteins that could otherwise promote tumor cell growth. DISCUSSION This study has identified Lin28B, a let-7 miRNA inhibitor, as a target of Merlin/NF2 that is important for the latter’s tumor-suppressive activity. Although the oncogenic and cell reprogram-

ming properties of Lin28 proteins are well defined (Shyh-Chang and Daley, 2013; Thornton and Gregory, 2012), exactly how Lin28B is regulated in response to cellular signaling has been difficult to determine. It is known that Musashi1 and SET7/9 enhance the localization of Lin28 in the nucleolus and inhibit the nuclear cropping of let-7 family miRNAs during early neural differentiation (Kawahara et al., 2011; Kim et al., 2014). Here, we demonstrate that Lin28B is regulated by a signaling mechanism that is activated by cell-cell contact. Specifically, we propose that Merlin/NF2 promotes let-7 miRNA maturation and reduces cell growth by suppressing nuclear/nucleolar Lin28B localization in a cell-contact-dependent manner (Figure 7F). In the absence of cell-cell contact, phosphorylated Merlin/NF2


(which has low affinity for Lin28B) is localized in the cytoplasm and nucleus. Accordingly, Lin28B localizes freely in both the nucleus/nucleolus and cytoplasm (Figures 4A, 4D, and S2A–S2C). Nuclear/nucleolar Lin28B then decreases the maturation of prilet-7 miRNAs, thereby allowing an accumulation of proteins promoting cell growth. On the other hand, following cell-cell contact, the non-phosphorylated form of Merlin/NF2 binds to Lin28B and sequesters it in the cytoplasm and plasma membrane. Lin28B is thus excluded from the nucleus/nucleolus, leading to an increase in mlet-7 miRNAs, a reduction in growth-promoting proteins, and a dampening of cell proliferation. Our report thus provides the first evidence of Merlin/NF2-dependent regulation of Lin28B in response to contact inhibition and describes the effects of that regulation. Although we observed that the subcellular localization of Lin28B correlated with the phosphorylation status of Merlin/ NF2, the underlying mechanism by which cell contact-mediated signaling controls Merlin/NF2 phosphorylation and localization remains to be dissected. Consistent with previous reports (Morrison et al., 2001; Okada et al., 2005), we observed that cell-contact-mediated signaling triggered dephosphorylation of Merlin/ NF2 at S518, but whether it is indeed this form of Merlin/NF2 that inhibits cell growth is not yet established. Conflicting conclusions regarding the phosphorylation-related localization of Merlin/NF2 have been reported in previous studies. For example, Li et al. described a form of Merlin/NF2 that was underphosphorylated at S518 and accumulated in the nucleus. This Merlin/NF2 isoform was found to suppress tumorigenesis by blocking the CRLDCAF1-mediated proteolysis pathway in human cancer cell lines (Li et al., 2010). In contrast, several other groups have reported that Merlin/NF2 phosphorylated at S518 is enriched in either the nucleus or cytoplasm depending on the cell type and cell-cycle stage (Guerrero et al., 2015; Muranen et al., 2005). It remains possible that the nuclear localization of Merlin/NF2 is regulated by a mechanism other than phosphorylation during contact inhibition, a subject currently under investigation. It has been previously reported that Lin28B is also a direct downstream target of mlet-7 (Guo et al., 2006). In our study, we observed that an increase or decrease in Lin28B consistently correlated with knockdown or overexpression of let-7, respectively (Figures S7A–S7D). Thus, alterations to Lin28B imposed by loss or gain of Merlin/NF2 function may also depend on the level of mlet-7. The usual pathways modulating protein stability do not appear to be involved, since treatment of H1299 cells with either MG132 (proteasome inhibitor) or bafilomycin (lysosome inhibitor) does not affect Lin28B protein levels (unpublished data). Further studies are needed to evaluate whether Merlin/NF2 deficiency is associated with mlet-7 reduction and/ or Lin28B accumulation in human tumors. Recent work has shown that inactivation of the Hippo pathway, or activation of its effectors YAP1/TAZ, inhibits the miRNA microprocessor machinery and so causes widespread miRNA suppression (Mori et al., 2014). Nevertheless, in our study, knockdown of YAP1/TAZ failed to rescue the expression of the mlet-7 family in Merlin/NF2-depleted H1299 cells (Figures 2E and 2F). Lin28 proteins have a direct effect on mlet-7 biogenesis, whereas YAP1 sequesters the microprocessor accessory

protein p72 and so may be a global suppressor of miRNAs. One possible explanation for this discrepancy is that Lin28B may have a stronger impact on mlet-7 biogenesis than does YAP1 in Merlin/NF2-depleted cells. YAP1 may be able to support mlet-7 biogenesis in the absence of Lin28, since inhibition of YAP1 enhances mlet7-dependent microprocessor reporter activity in HaCaT cells (Mori et al., 2014). In fact, we could not detect expression of Lin28 proteins in these cells in our preliminary immunoblot experiment (data not shown). However, we cannot rule out another possibility: that the microprocessor machinery complex in H1299 cells does not utilize p72 but rather p68, which is functionally related to p72 but free from YAP-mediated sequestration. In any case, our results indicate that Lin28B and YAP1/TAZ are coordinately in charge of promoting the growth of Merlin/NF2deficient H1299 cells, because both proteins are necessary for sphere formation following Merlin/NF2 depletion. Our data also raise the possibility that malignant Merlin/NF2-related tumors may exhibit both YAP1/TAZ activation and Lin28 accumulation, a scenario supported by our observation that both occur in H290 cells, a human Merlin/NF2-deficient malignant mesothelioma cell line. Since Merlin/NF2-deficient mice are clearly predisposed to developing cancers in multiple tissues (McClatchey et al., 1998), it will be interesting to analyze the contribution of the Merlin/NF2-Lin28-let-7 axis, in addition to the canonical Merlin/NF2-MST-LATS-YAP1 axis, to the prevention of tumor development in mouse models. The results of such studies could point to a therapeutic approach that targets the Merlin/NF2Lin28-let-7 axis and might be beneficial for the treatment of Merlin/NF2-related tumors in humans. EXPERIMENTAL PROCEDURES Cell Culture, Transfection, and Sphere Formation HEK293T, Huh7, HeLa cells (JCRB), and Caco2 (ATCC) cells were maintained in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS; Hyclone) and were transfected using linear polyethylenimine (MW 25,000; Polysciences). H1299 cells (ATCC) and H290 cells (a kind gift of Dr. A.F. Gazdar, University of Texas Southwestern Medical Center, Dallas, TX) were cultured in RPMI1640 (Nacalai) supplemented with 10% FBS. For analyses of cell-density-dependent responses, cells were cultured at low (2 3 105/9.6cm2) or high (1.5 3 106/9.6cm2) cell density for 3 days. For sphere formation, H1299 cells infected with lentivirus expressing specific shRNAs were dissociated with trypsin-EDTA solution (Nacalai) and filtered through a cell strainer (40-mm pores; BD Falcon). The resulting single-cell suspensions were plated at 1,000 cells/ml in serum-free DMEM-F12 (Wako) containing 10 ng/ml basic fibroblast growth factor (bFGF; R&D Systems), 20 ng/ml EGF (Sigma), 5 ng/ml insulin (Nacalai), and 0.4% BSA (Sigma). Gene expression in the inducible H290 cell line was initiated by adding Dox (AppliChem) to a final concentration of 100 ng/ml in the culture medium. Plasmids pCSC-flagMerlin/NF2, pCS2-mycLin28B, pCS2-flagLin28B, and pCS2flagMST1 were generated by PCR subcloning from cDNAs synthesized using HEK293T total RNA. Point and deletion mutants of Merlin/NF2 and Lin28B constructs were generated using single-primer-based site-directed mutagenesis as described previously (Hikasa et al., 2010). A PCR fragment of flagMerSA was subcloned into a Dox-inducible entry vector and then flipped into the SLIK-neo vector using Gateway LR Clonase II Enzyme mix (Invitrogen). Cloning and mutagenesis were verified by standard DNA sequencing. Primer sequences for mutagenesis are listed in Supplemental Experimental Procedures.


Protein Identification by Mass Spectrometry Lysates of HEK293T cells expressing flagMerWT were incubated with antiflag (M2)-conjugated agarose beads (Sigma) and fractionated by standard SDS-PAGE. Gels were sliced into pieces, stained with silver (Sigma), and digested with Trypsin Gold Mass Spectrometry grade (Promega). Digests were analyzed with a nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) system employing an LTQ ion trap mass spectrometer (Thermo Scientific). Initial identification of proteins from LC-MS/MS data was performed using the Mascot search engine (Matrix Science). Any protein for which peptides were also found in negative control lysates of HEK293T cells was discarded. Immunocytochemistry H1299 and H290 cells were fixed with 4% paraformaldehyde for 5 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 3 min at RT, blocked with 3% FBS, and incubated at 4 C overnight with Ab against Lin28B (1:200, D4H1, #11965; Cell Signaling), Merlin/NF2 (1:200, #12896; Cell Signaling) or B23 (1:500, B0556; Sigma). For double staining to detect BrdU incorporation plus Lin28B protein, H290MerSA cells were treated with 1.5M HCl for 20 min prior to incubation with anti-Lin28B (1:200, D4H1, #11965; Cell Signaling) and anti-BrdU (1:200, #5292; Cell Signaling) Abs. After washing in PBS, cells were incubated for 3 hr with anti-rabbit Alexa 488 (1:1,000, A11008; Invitrogen) and/or anti-rabbit-Alexa Fluor 568 (1:1,000, A11031; Invitrogen) Abs. Nuclei were visualized by DAPI staining (Wako). Immunoprecipitation and Immunoblotting Cells were lysed in lysis buffer containing 0.5%–1% Triton X-100, 50 mM TrisHCl, 50-150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 10 mM NaF, and 1 mM Na3VO4. Supernatants were incubated at 4 C overnight with anti-flag-agarose beads (Sigma) or anti-Lin28B Ab (D4H1, #11965; Cell Signaling) plus Protein G-agarose beads (Nacalai). Antibody-bound beads were washed three times with lysis buffer, boiled in SDS-PAGE sample buffer, and subjected to standard immunoblotting. Primary Abs recognized were Lin28B, Merlin/NF2, Merlin/NF2 518S, pYAP1, YAP1, TAZ, a-tubulin, EGFR, IMP1 (all from Cell Signaling: #11965, #12896, #13281, #4911, #4912, #4883, #2144, #4267, and #8482, respectively); lamin (#16048; Abcam); 9E10 myc (sc-40; Santa Cruz Biotechnology), or flag (M2) epitope (#S1804; Sigma). Primary Abs were detected using horseradish peroxidase (HRP)-conjugated secondary Abs (Cell Signaling). Lentivirus-Mediated Gene Expression Lentiviral vectors (CS-RfA-ETBsd, pENTRE4-H1) were obtained from the RIKEN BRC DNA Bank and injected into HEK293T cells as described previously (Sasaki et al., 2011). Culture supernatants containing virus particles were subjected to ultracentrifugation at 50,000 3 g for 2 hr to obtain concentrated lentivirus preparations. Sequences of shRNAs expressed by lentiviral vectors are listed in Supplemental Experimental Procedures. Oligonucleotide Transfection Double-stranded oligoRNAs mimicking let-7g were obtained from Ambion (mirVana miRNA mimic for let-7g), while locked nucleic acids (LNAs) against let-7 miRNAs were obtained from Exiqon (hsa-let-7 miRCURY LNA microRNA family inhibitor). Either scramble siRNA or double-stranded oligoDNA was used as a negative control. Transfection of oligonucleotides (10 nM) into exponentially growing H1299 or H290 cells was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s protocol. miRNA Expression Analysis Total RNAs extracted with RNAiso (Takara) were subjected to reverse transcription using the TaqMan MicroRNA RT Kit (Applied Biosystems). TaqMan miRNA assays for U6 small nuclear RNA (snRNA), mlet-7a, 7c, 7g, and miR16 (Applied Biosystems) were used to quantify mature miRNAs. Expression of pri-miRNAs was analyzed by qPCR using TaqMan Universal Master Mix II (Applied Biosystems) with the TaqMan Pri-miRNA Assay for pri-let-7a, 7b, and 7g (Applied Biosystems) and the TaqMan Gene Expression Assay for actin (Applied Biosystems). All qPCR reactions were carried out using an ABI7500 real-time qPCR system. Data were normalized to U6 snRNA expression.

Subcellular Fractionation Nuclear, cytoplasmic, and plasma membrane fractions of cell lysates were prepared using FOCUS SubCel (G-Biosciences) according to the manufacturer’s instructions. Subcellular fractionation was validated by immunoblotting using anti-lamin (#16048; Abcam), anti-a-tubulin (#2144; Cell Signaling), and anti-EGFR (#4267; Cell Signaling) Abs to identify nuclear, cytoplasmic, and plasma membrane markers, respectively. RNA Immunoprecipitation RNA immunoprecipitation (RIP) assay was carried out using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. H1299 cells expressing shRNA or flagMer constructs were lysed and subjected to immunoblot analysis in order to equalize amounts of endogenous Lin28B protein. Equalized cell lysates were incubated for 4 hr at 4 C with anti-Lin28B Ab (5 mg; Cell Signaling) or control rabbit IgG (10 mg) conjugated to magnetic beads (50 ml). Mixtures of beads, immunoprecipitated proteins, and miRNAs were magnetically separated. Immunoprecipitated complexes were washed and treated with proteinase K. RNA was extracted using the phenol/chloroform method and converted to cDNA using the Transcriptor First Strand cDNA synthesis kit (Roche). cDNA was then subjected to qRT-PCR to detect pri-let-7 family member sequences. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.02.075. AUTHOR CONTRIBUTIONS Conception and design, H.H. and A.S.; provision of key materials, Y.S.; acquisition of data, H.H.; analysis and interpretation of data, H.H., Y.S., and A.S.; and writing of the manuscript, H.H. and A.S. All authors have reviewed the manuscript and approved its contents. ACKNOWLEDGMENTS We are grateful to Keiji Ito for critical reading of the manuscript. We also thank Masanori Taira, Keiji Ito, Koichi Kawahara, Nishio Miki, and members of the Suzuki laboratory for helpful experimental discussions. This work was supported by grants from the Ministry of Education, Culture, Sports and Technology of Japan (MEXT) and the Japan Society for the Promotion of Science (JSPS) (grant 15K07052 to H.H. and grants 26114005 and 24240120 to A.S.); the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University; P-DIRECT (to A.S.); the Uehara Memorial Foundation (to A.S.); the Takeda Medical Foundation (to H.H. and A.S.); and the Shinnihon Foundation of Advanced Medical Treatment Research (to A.S.). Received: August 3, 2015 Revised: December 20, 2015 Accepted: February 18, 2016 Published: March 17, 2016 REFERENCES Bai, Y., Liu, Y.J., Wang, H., Xu, Y., Stamenkovic, I., and Yu, Q. (2007). Inhibition of the hyaluronan-CD44 interaction by merlin contributes to the tumor-suppressor activity of merlin. Oncogene 26, 836–850. Benhamouche, S., Curto, M., Saotome, I., Gladden, A.B., Liu, C.H., Giovannini, M., and McClatchey, A.I. (2010). Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver. Genes Dev. 24, 1718–1730. Cho, J., Chang, H., Kwon, S.C., Kim, B., Kim, Y., Choe, J., Ha, M., Kim, Y.K., and Kim, V.N. (2012). LIN28A is a suppressor of ER-associated translation in embryonic stem cells. Cell 151, 765–777.


Curto, M., Cole, B.K., Lallemand, D., Liu, C.H., and McClatchey, A.I. (2007). Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. J. Cell Biol. 177, 893–903.

Madison, B.B., Liu, Q., Zhong, X., Hahn, C.M., Lin, N., Emmett, M.J., Stanger, B.Z., Lee, J.S., and Rustgi, A.K. (2013). LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7. Genes Dev. 27, 2233–2245.

Graf, R., Munschauer, M., Mastrobuoni, G., Mayr, F., Heinemann, U., Kempa, S., Rajewsky, N., and Landthaler, M. (2013). Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation. RNA Biol. 10, 1146–1159.

Mayr, F., and Heinemann, U. (2013). Mechanisms of Lin28-mediated miRNA and mRNA regulation–a structural and functional perspective. Int. J. Mol. Sci. 14, 16532–16553.

Guerrero, P.A., Yin, W., Camacho, L., and Marchetti, D. (2015). Oncogenic role of Merlin/NF2 in glioblastoma. Oncogene 34, 2621–2630. Guo, Y., Chen, Y., Ito, H., Watanabe, A., Ge, X., Kodama, T., and Aburatani, H. (2006). Identification and characterization of lin-28 homolog B (LIN28B) in human hepatocellular carcinoma. Gene 384, 51–61. Hagan, J.P., Piskounova, E., and Gregory, R.I. (2009). Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat. Struct. Mol. Biol. 16, 1021–1025. Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646–674. Heo, I., Joo, C., Cho, J., Ha, M., Han, J., and Kim, V.N. (2008). Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol. Cell 32, 276–284. Heo, I., Joo, C., Kim, Y.K., Ha, M., Yoon, M.J., Cho, J., Yeom, K.H., Han, J., and Kim, V.N. (2009). TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell 138, 696–708. Hikasa, H., Ezan, J., Itoh, K., Li, X., Klymkowsky, M.W., and Sokol, S.Y. (2010). Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification. Dev. Cell 19, 521–532. Hong, W., and Guan, K.L. (2012). The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway. Semin. Cell Dev. Biol. 23, 785–793. Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A., Labourier, E., Reinert, K.L., Brown, D., and Slack, F.J. (2005). RAS is regulated by the let-7 microRNA family. Cell 120, 635–647. Kawahara, H., Okada, Y., Imai, T., Iwanami, A., Mischel, P.S., and Okano, H. (2011). Musashi1 cooperates in abnormal cell lineage protein 28 (Lin28)-mediated let-7 family microRNA biogenesis in early neural differentiation. J. Biol. Chem. 286, 16121–16130. Kim, S.K., Lee, H., Han, K., Kim, S.C., Choi, Y., Park, S.W., Bak, G., Lee, Y., Choi, J.K., Kim, T.K., et al. (2014). SET7/9 methylation of the pluripotency factor LIN28A is a nucleolar localization mechanism that blocks let-7 biogenesis in human ESCs. Cell Stem Cell 15, 735–749. Kissil, J.L., Wilker, E.W., Johnson, K.C., Eckman, M.S., Yaffe, M.B., and Jacks, T. (2003). Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1. Mol. Cell 12, 841–849. Kumar, M.S., Lu, J., Mercer, K.L., Golub, T.R., and Jacks, T. (2007). Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39, 673–677. Lee, Y.S., and Dutta, A. (2007). The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes Dev. 21, 1025–1030. Lehrbach, N.J., Armisen, J., Lightfoot, H.L., Murfitt, K.J., Bugaut, A., Balasubramanian, S., and Miska, E.A. (2009). LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 16, 1016–1020. Li, W., You, L., Cooper, J., Schiavon, G., Pepe-Caprio, A., Zhou, L., Ishii, R., Giovannini, M., Hanemann, C.O., Long, S.B., et al. (2010). Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. Cell 140, 477–490. Li, W., Cooper, J., Karajannis, M.A., and Giancotti, F.G. (2012). Merlin: a tumour suppressor with functions at the cell cortex and in the nucleus. EMBO Rep. 13, 204–215. Li, W., Cooper, J., Zhou, L., Yang, C., Erdjument-Bromage, H., Zagzag, D., Snuderl, M., Ladanyi, M., Hanemann, C.O., Zhou, P., et al. (2014). Merlin/ NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus. Cancer Cell 26, 48–60.

McClatchey, A.I., Saotome, I., Mercer, K., Crowley, D., Gusella, J.F., Bronson, R.T., and Jacks, T. (1998). Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev. 12, 1121–1133. Molenaar, J.J., Domingo-Ferna´ndez, R., Ebus, M.E., Lindner, S., Koster, J., Drabek, K., Mestdagh, P., van Sluis, P., Valentijn, L.J., van Nes, J., et al. (2012). LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression. Nat. Genet. 44, 1199–1206. Mori, M., Triboulet, R., Mohseni, M., Schlegelmilch, K., Shrestha, K., Camargo, F.D., and Gregory, R.I. (2014). Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer. Cell 156, 893–906. Morrison, H., Sherman, L.S., Legg, J., Banine, F., Isacke, C., Haipek, C.A., Gutmann, D.H., Ponta, H., and Herrlich, P. (2001). The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44. Genes Dev. 15, 968–980. Muranen, T., Gro¨nholm, M., Renkema, G.H., and Carpe´n, O. (2005). Cell cycledependent nucleocytoplasmic shuttling of the neurofibromatosis 2 tumour suppressor merlin. Oncogene 24, 1150–1158. Nam, Y., Chen, C., Gregory, R.I., Chou, J.J., and Sliz, P. (2011). Molecular basis for interaction of let-7 microRNAs with Lin28. Cell 147, 1080–1091. Nguyen, L.H., Robinton, D.A., Seligson, M.T., Wu, L., Li, L., Rakheja, D., Comerford, S.A., Ramezani, S., Sun, X., Parikh, M.S., et al. (2014). Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models. Cancer Cell 26, 248–261. Nishino, J., Kim, I., Chada, K., and Morrison, S.J. (2008). Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell 135, 227–239. Okada, T., Lopez-Lago, M., and Giancotti, F.G. (2005). Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane. J. Cell Biol. 171, 361–371. Pellock, B.J., Buff, E., White, K., and Hariharan, I.K. (2007). The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling. Dev. Biol. 304, 102–115. Piskounova, E., Viswanathan, S.R., Janas, M., LaPierre, R.J., Daley, G.Q., Sliz, P., and Gregory, R.I. (2008). Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J. Biol. Chem. 283, 21310–21314. Piskounova, E., Polytarchou, C., Thornton, J.E., LaPierre, R.J., Pothoulakis, C., Hagan, J.P., Iliopoulos, D., and Gregory, R.I. (2011). Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms. Cell 147, 1066– 1079. Rehfeld, F., Rohde, A.M., Nguyen, D.T., and Wulczyn, F.G. (2015). Lin28 and let-7: ancient milestones on the road from pluripotency to neurogenesis. Cell Tissue Res. 359, 145–160. Roush, S., and Slack, F.J. (2008). The let-7 family of microRNAs. Trends Cell Biol. 18, 505–516. Sasaki, M., Kawahara, K., Nishio, M., Mimori, K., Kogo, R., Hamada, K., Itoh, B., Wang, J., Komatsu, Y., Yang, Y.R., et al. (2011). Regulation of the MDM2P53 pathway and tumor growth by PICT1 via nucleolar RPL11. Nat. Med. 17, 944–951. Sekido, Y. (2013). Molecular pathogenesis of malignant mesothelioma. Carcinogenesis 34, 1413–1419. Shyh-Chang, N., and Daley, G.Q. (2013). Lin28: primal regulator of growth and metabolism in stem cells. Cell Stem Cell 12, 395–406. Shyh-Chang, N., Zhu, H., Yvanka de Soysa, T., Shinoda, G., Seligson, M.T., Tsanov, K.M., Nguyen, L., Asara, J.M., Cantley, L.C., and Daley, G.Q.


(2013). Lin28 enhances tissue repair by reprogramming cellular metabolism. Cell 155, 778–792. Thornton, J.E., and Gregory, R.I. (2012). How does Lin28 let-7 control development and disease? Trends Cell Biol. 22, 474–482. Van Wynsberghe, P.M., Kai, Z.S., Massirer, K.B., Burton, V.H., Yeo, G.W., and Pasquinelli, A.E. (2011). LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 18, 302–308.

A tight junction-associated Merlin-angiomotin complex mediates Merlin’s regulation of mitogenic signaling and tumor suppressive functions. Cancer Cell 19, 527–540. Yu, J., Zheng, Y., Dong, J., Klusza, S., Deng, W.M., and Pan, D. (2010). Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded. Dev. Cell 18, 288–299.

Viswanathan, S.R., and Daley, G.Q. (2010). Lin28: A microRNA regulator with a macro role. Cell 140, 445–449.

Zhang, N., Bai, H., David, K.K., Dong, J., Zheng, Y., Cai, J., Giovannini, M., Liu, P., Anders, R.A., and Pan, D. (2010). The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev. Cell 19, 27–38.

Viswanathan, S.R., Daley, G.Q., and Gregory, R.I. (2008). Selective blockade of microRNA processing by Lin28. Science 320, 97–100.

Zhou, J., Ng, S.B., and Chng, W.J. (2013). LIN28/LIN28B: an emerging oncogenic driver in cancer stem cells. Int. J. Biochem. Cell Biol. 45, 973–978.

Wilbert, M.L., Huelga, S.C., Kapeli, K., Stark, T.J., Liang, T.Y., Chen, S.X., Yan, B.Y., Nathanson, J.L., Hutt, K.R., Lovci, M.T., et al. (2012). LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance. Mol. Cell 48, 195–206.

Zhu, H., Shah, S., Shyh-Chang, N., Shinoda, G., Einhorn, W.S., Viswanathan, S.R., Takeuchi, A., Grasemann, C., Rinn, J.L., Lopez, M.F., et al. (2010). Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies. Nat. Genet. 42, 626–630.

Xiao, G.H., Beeser, A., Chernoff, J., and Testa, J.R. (2002). p21-activated kinase links Rac/Cdc42 signaling to merlin. J. Biol. Chem. 277, 883–886.

Zhu, H., Shyh-Chang, N., Segre`, A.V., Shinoda, G., Shah, S.P., Einhorn, W.S., Takeuchi, A., Engreitz, J.M., Hagan, J.P., Kharas, M.G., et al.; DIAGRAM Consortium; MAGIC Investigators (2011). The Lin28/let-7 axis regulates glucose metabolism. Cell 147, 81–94.

Yi, C., Troutman, S., Fera, D., Stemmer-Rachamimov, A., Avila, J.L., Christian, N., Persson, N.L., Shimono, A., Speicher, D.W., Marmorstein, R., et al. (2011).
