Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

Article

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway Graphical Abstract

Authors Sayan Chakraborty, Kizito Njah, Ajaybabu V. Pobbati, ..., Vinay Tergaonkar, Chwee Teck Lim, Wanjin Hong

Correspondence [email protected] (S.C.), [email protected] (W.H.)

In Brief Chakraborty et al. report that the extracellular matrix protein Agrin is a mechanotransducing signal activating YAP through the integrin-focal adhesionLrp4/MuSK receptor pathway. Agrin signals matrix and cellular rigidity by activating FAK-ILK-PAK1 signaling that negates the Hippo tumor-suppressor pathway. Importantly, Agrin relies on YAP for oncogenic activities underlying liver cancer.

Highlights d

Agrin mechanoactivates YAP by negating the Hippo pathway

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Agrin transduces matrix rigidity signals to YAP through an integrin-Lrp4/MuSK pathway

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Agrin-induced FAK-ILK-PAK1 restricts focal adhesion enrichment of Hippo signaling

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The Agrin-YAP mechanotransduction network cooperates in liver cancer development

Chakraborty et al., 2017, Cell Reports 18, 2464–2479 March 7, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.02.041

Cell Reports

Article Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway Sayan Chakraborty,1,* Kizito Njah,1 Ajaybabu V. Pobbati,1 Ying Bena Lim,2 Anandhkumar Raju,1 Manikandan Lakshmanan,1 Vinay Tergaonkar,1 Chwee Teck Lim,2,3,4 and Wanjin Hong1,5,* 1Institute

of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore 2Infectious Diseases Interdisciplinary Research, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, Singapore 138602, Singapore 3Department of Biomedical Engineering, National University of Singapore, 4 Engineering Drive 3, Singapore 117583, Singapore 4Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore 5Lead Contact *Correspondence: [email protected] (S.C.), [email protected] (W.H.) http://dx.doi.org/10.1016/j.celrep.2017.02.041

SUMMARY

The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM. INTRODUCTION The extracellular matrix (ECM), a non-cellular component of the tissue microenvironment, dictates cell behavior through mechanical changes during embryogenesis and cancer (Butcher et al., 2009; DuFort et al., 2011; Hoffman et al., 2011; Levental et al., 2009). Cells sense mechanical changes in the ECM and translate them into defined signaling responses, a process termed mechanotransduction. The transcriptional coactivators Yes-associated protein (YAP) and TAZ (transcriptional activator with PDZ binding motif) act as nuclear effectors to changes in ECM stiffness and cell architecture as well as cytoskeletal alterations (Aragona et al., 2013; Dupont et al., 2011; Halder et al., 2012). YAP and TAZ are also the

converging effectors of the Hippo pathway that regulates growth and organ size in flies and mammals (Huang et al., 2005; Piccolo et al., 2014; Zeng and Hong, 2008; Zhao et al., 2011b). The mammalian Hippo pathway comprises two core kinases (Mst1/2 and LATS1/2) and adaptor proteins, respectively. When activated by an upstream signal, the four-point-one, ezrin, radixin, moesin (FERM)-domain-containing tumor-suppressor protein, Merlin, activates the core kinases (Hamaratoglu et al., 2006; Zhang et al., 2010). Activated LATS1/2 inhibits YAP functions through phosphorylations at serine (Ser)127 residue, leading to its cytosolic sequestration and at Ser381, priming it for proteasomal degradation (Dong et al., 2007; Zhao et al., 2007, 2010, 2011b). Hence, various signaling cascades hijack the Hippo pathway to activate YAP/TAZ in cancer (Azzolin et al., 2012; Fan et al., 2013; Harvey et al., 2013; Serrano et al., 2013; Yu et al., 2012). Mechanotransduction related to cell shape and cytoskeletal changes either affect YAP independently of Hippo signaling (Aragona et al., 2013; Dupont et al., 2011) or may involve LATS/Hippo kinases (Heidary Arash et al., 2014; Sansores-Garcia et al., 2011; Wada et al., 2011). The integrin-focal adhesion (FA) complex acts as the central mechanosensor detecting ECM changes in response to soluble ECM factors (Butcher et al., 2009; Humphrey et al., 2014; Ross et al., 2013). Therefore, signaling downstream of integrins (including integrin-linked kinase [ILK], focal adhesion kinase [FAK], Src, and b-Pix) are known to regulate YAP and TAZ (Heidary Arash et al., 2014; Kim and Gumbiner, 2015; Serrano et al., 2013). Although these biomechanical changes are actively sensed to regulate YAP/TAZ, the identity and role(s) of soluble ECM factors (mechanotransduction signals) and their nexus with the integrin pathway(s) to relay mechanical signals remain unclear. As an ECM proteoglycan, Agrin binds to its co-receptor(s) lipoprotein-related receptor-4 (Lrp4) and muscle-specific kinase (MuSK) to maintain functional neuromuscular junctions (Chang et al., 2016; Kim et al., 2008; McMahan, 1990). Besides its canonical function at the neuromuscular junctions, Agrin expressed in liver cancer cells sustain focal adhesion integrity and drives hepatocellular carcinoma (HCC) through the Lrp4/MuSK pathway (Chakraborty et al., 2015). Since YAP is

2464 Cell Reports 18, 2464–2479, March 7, 2017 ª 2017 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. Loss of Agrin Function Inactivates YAP (A) Western blot in control or Agrin depleted cell lines detecting YAP phosphorylation and b-actin as loading control. Phospho-YAP levels normalized to YAP were determined using ImageJ. (B) Indicated cell lines were treated with IgG or indicated concentrations of Agrin antibody. MTS [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay was done after 5 days and represented as (%) inhibition of growth compared to IgG (n = 3, Student’s t test, **p < 0.005). (C) Indicated cells were treated as in (B) with 10 mg/mL antibody for 2 days and western blotted for indicated proteins. b-Actin served as a loading control. (D) Western blot analysis in control or Agrin antibody treated tumors. b-Actin served as a loading control. Densitometric analysis of phospho-YAP was normalized to YAP levels (n = 3, mean ± SD, **p < 0.005).

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also activated in liver cancer through perturbation of the Hippo pathway (Dong et al., 2007; Lu et al., 2010; Yimlamai et al., 2015), whether Agrin crosstalks within YAP-mechanotransduction network by inactivating the Hippo pathway is an open question. Herein, we identify a plausible mechanotransduction link between Agrin and YAP that cooperate in liver cells during hepatocarcinogenesis. Importantly, Agrin’s oncogenic role depends on the transcriptional and mechanoactivity of YAP and therefore coupling ECM-mediated mechanotransduction and the Hippo pathway. RESULTS Inhibiting Agrin Leads to YAP Inactivation Perturbation of the Hippo pathway is associated with liver overgrowth and cancer (Dong et al., 2007; Yimlamai et al., 2015). As Agrin plays a critical role in liver tumorigenesis (Chakraborty et al., 2015), we therefore explored whether Agrin signaling inactivates the Hippo pathway. To this end, we investigated whether Agrin depletion affects the phosphorylation status of YAP, a key event in YAP regulation by the Hippo pathway. Agrin depletion in HCC and breast cancer MCF7 (with high YAP activity) cells increased YAP phosphorylation at Ser127 residue (Figures 1A and S1A). Furthermore, an Agrin-function-blocking antibody that inhibited cell growth in a dose-responsive manner also enhanced YAP phosphorylation (Figures 1B and 1C). In vivo, the xenograft tumors treated with this antibody revealed higher YAP phosphorylation as compared to the PBS-treated controls (Figure 1D). Consistent with increased YAP phosphorylation, YAP was shifted from the nucleus to the cytoplasm in Agrindepleted cells (regardless of the cell density) (Figure 1E). Although high cell density robustly increased YAP phosphorylation, it had negligible effects on Agrin protein levels (Figure S1B). Importantly, the nuclear localization of YAP was restored in knockdown cells by expressing rat-Agrin (Agrin rescued cells) resistant to small hairpin RNA (shRNA) (Chakraborty et al., 2015) (Figure 1E). The similar nuclear-cytoplasmic shift of TAZ was also observed upon Agrin depletion (Figure S1C). Blocking nuclear export by leptomycin B rescued the nuclear YAP levels in Agrin knockdown cells, confirming that Agrin depletion shifts YAP from the nucleus to the cytoplasm (Figure S1D).

Multiple isoforms of Agrin exist naturally. However, only neuronal forms contain 8-amino-acid inserts within Z exons, commonly referred as Agrin-Z8, that potently activate Lrp4/MuSK and cluster acetylcholine receptors (Cox and Erler, 2011; Kim et al., 2008). Therefore, we probed which Agrin isoform is predominant in HCC cells. Similar to neuroblastoma SK-N-SH cells, serving as a positive control, Agrin (Z8) was expressed in HCC cells but was absent in rhabdomyosarcoma cell line RH30 (Figure S1E). Moreover, secretory but not transmembrane isoform of Agrin was expressed in HCC cells (Figure S1F). Hence, we analyzed whether supplementing soluble neural or non-neural (without Z8 insert) Agrin activates YAP by dephosphorylation. This was performed in minor histocompatibility antigen bearing normal liver cell line (MIHA) cells that have low endogenous Agrin coupled to higher phosphorylated YAP when compared to Hep3B cells (Figure S1G). Interestingly, both neural and non-neural Agrin (Agrin) dephosphorylated YAP in a dose-responsive manner (Figure S1H). Treatment with neural and non-neural Agrin proteins reduced YAP phosphorylation even in Agrin-depleted cells (Figure 1F), thereby suggesting that both forms of Agrin may regulate YAP. These data indicate that Agrin depletion inactivates YAP by increasing its inhibitory phosphorylation and cytoplasmic sequestration. However, this can be rescued by exogenous neural and non-neural Agrin. Agrin Stabilizes YAP by Inhibiting Interactions with Amot-14-3-3 and Increases Expression of YAP Targets The Hippo pathway mediated phosphorylation of YAP creates a 14-3-3 binding site, leading to cytoplasmic sequestration of YAP by interaction with 14-3-3 proteins (Basu et al., 2003; Zhao et al., 2007). In addition, angiomotin proteins, including Amot-p130, interact with YAP in the cytoplasm independent of 14-3-3 (Chan et al., 2011, 2013; Zhao et al., 2011a). Indeed, increased association of YAP with Amot-p130 and 14-3-3 (epsilon ‘‘ε’’ subunit) was seen in Agrin-depleted HCC cells (Figure 1G). Accumulation of YAP in the cytoplasm of Agrin depleted cells accounted for its robust association with Amot-p130 (Figure 1H). Likewise, there was increased cytoplasmic colocalization between YAP and 14-3-3ε upon Agrin depletion (Figure S1I). Neural Agrin enhanced nuclear localization of YAP with a corresponding decrease in the colocalization with 14-3-3ε in a dose and

(E) Immunofluorescence assay for YAP in control, Agrin-depleted, and Agrin-rescued cells. 150–200 cells were analyzed. C > N and N > C refers to predominant cytoplasmic and nuclear YAP, respectively. Scale bar, 10 mm. (F) Control or Agrin-depleted MHCC-LM3 cells were either left untreated or treated with neural or non-neural Agrin for a day and western blotted for indicated proteins. b-Actin served as a loading control. (G) Control or Agrin knockdown cell lysates were immunoprecipitated with YAP antibody and western blotted for 14-3-3ε and Amot-p130. The blots were stripped and probed for YAP. 10% cell lysates were used as input. The fraction (%) of Amot or 14-3-3 interacting with YAP is shown. (H) Nuclear and cytosolic fractions from control and Agrin-depleted MHCC-LM3 cells were verified by western blot (left) for nuclear (lamin A) and cytoplasmic (RhoGDI) markers, respectively. They were immunoprecipitated by YAP antibody and western blotted for Amot (right). The blot was re-probed for YAP. (I) Control and Agrin-depleted MHCC-LM3 cells were treated with 30 mg/mL CHX for the indicated time points and western blotted for YAP. Relative YAP levels normalized to b-actin are shown. Relative YAP levels were analyzed as above. (J) Cells from (I) were treated with CHX alone or in combination with 10 mM MG132 for 6 hr and analyzed by western blot. Relative YAP levels were analyzed as above. (K) MIHA cell lysates, either left untreated or treated with 5 mg/mL Agrin for 1 day, were analyzed by western blot. Low (l.e) or high (h.e) YAP exposure is shown and quantified as in (I). (L) Control and Agrin-depleted MHCC-LM3 cell lysates were immunoprecipitated with YAP antibody and western blotted for TEAD4. The blots were re-probed for YAP. 10% lysates were used as input. (M) Western blot analysis in control and Agrin-depleted MHCC-LM3 cells for indicated proteins. Reduction in expression (%) normalized to b-actin is shown. See also Figure S1.

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time-dependent manner in Agrin-depleted cells (Figures S1J and S1K). The phosphorylation of cytosolic YAP at Ser381 residue that primes it toward proteasomal degradation, was enhanced upon Agrin knockdown (Figure S1L). Agrin depletion did not affect YAP mRNA levels (Figure S1M) but reduced YAP protein upon cycloheximide (CHX) treatment for 4–6 hr (Figure 1I). Treatment with proteasomal inhibitor MG132 along with CHX robustly rescued YAP levels in Agrin knockdown cells while only having a partial effect in control cells (Figure 1J). Moreover, cytoplasmic YAP in Agrin-depleted cells did not localize to lysosomes, thereby suggesting that YAP in Agrin-depleted cells is likely degraded within proteasomes, but not in lysosomes (Figure S1N). Conversely, neural Agrin enhanced YAP levels and delayed its degradation in the presence of CHX (Figure 1K). Together, depletion of Agrin inactivates YAP by (1) shifting it to the cytoplasm, where it is sequestered by Amot and 14-3-3 proteins, and (2) excessive YAP phosphorylation, which triggers its proteasomal degradation, thereby destabilizing YAP. Since YAP binds TEA-domain-containing transcriptional factors (TEAD1–4) to transcribe its target genes (Hong and Guan, 2012), this interaction between YAP and TEAD4 was severely compromised by Agrin knockdown (Figure 1L). As a result, several endogenous YAP targets (IGFBP3, Axl, Cyr61, connective tissue growth factor [CTGF], and Jagged-1) were also significantly downregulated upon Agrin depletion (Figure 1M). Collectively, these findings suggest that Agrin stabilizes nuclear YAP accumulation, interaction with TEADs, and the expression of YAP targets. Agrin Promotes the Mechanoresponsiveness of YAP YAP/TAZ acts as nuclear relay of mechanical cues sensed by cells from their ECM (Dupont et al., 2011; Halder et al., 2012). We, therefore, tested the possibility of a functional link between Agrin and the mechanosensing pathway to YAP/TAZ by utilizing stiffness manipulated hydrogel substrates (Fischer et al., 2012) (Figure S2A). Interestingly, cells responded to stiff ECM by increasing Agrin expression and formed an intercalating meshlike network with collagen I that was reduced in soft ECM (Figures 2A and S2B). Increased YAP phosphorylation and enhanced cytoplasmic distribution were evident in cells on soft gels (Figures 2A and S2C) (Aragona et al., 2013; Dupont et al., 2011). Enhanced interaction between YAP and 14-3-3ε in cells grown in compliant versus hard substrates further verified YAP’s mechanoactivity toward ECM stiffness (Figure S2D). Interestingly, Agrin knockdown in cells cultured in stiff ECM shifted nuclear YAP to the cytosol, regardless of cell density (Figure 2B). In contrast, cells in softer ECM were circular with cytoplasmic YAP (Figure S2E, second panel, and Figure 2C, first panel). Supplementing neural Agrin in soft ECM changed the cell morphology (Figure S2E) and induced nuclear localization of YAP in a dose-dependent fashion (Figure 2C). Further, Agrin depletion retained YAP in the cytoplasm and reduced growth of MHCC-LM3 cells in a three-dimensional (3D) spheroid matrix stiffness model that mimicked ‘‘hard’’ ECM (Aragona et al., 2013) (Figure 2D). In contrast, spheroids enclosed in ‘‘soft’’ ECM were inherently smaller with cytoplasmic YAP (Figures 2E and S2F). Supplementing Agrin over a period of 6 days significantly increased sphere sizes with nuclear localization of YAP even in soft ECM (Figures 2E and S2F). These results suggest that

exogenous Agrin is sufficient to activate cellular YAP in soft ECM, thereby mimicking the effects of stiff ECM. As knockdown of Agrin in hard ECM shifted YAP to the cytosol, these results together suggest that Agrin is both necessary (in hard ECM) and sufficient (when replenished in soft ECM) to sustain nuclear YAP. Interestingly, this effect was specific to Agrin, because knockdown of perlecan (an Agrin-related proteoglycan), affected neither YAP phosphorylation nor YAP localization (Figure S2G). As YAP localization varies under altered cell geometry (Dupont et al., 2011), nuclear YAP in cells confined to large square fibronectin patterns was strikingly shifted to cytoplasm upon Agrin depletion (Figures 2F and 2G, left). Nuclear YAP localization was rescued by supplementing Agrin in
70% of knockdown cells (Figures 2F and 2G, left). In contrast, cells geometrically confined to small ‘‘islands’’ retain YAP in the cytoplasm (Dupont et al., 2011). Exogenous Agrin partially stimulated nuclear localization of YAP even in the cells confined to small areas (Figures 2F and 2G, right), suggesting that Agrin is critical for YAP’s localization upon altering cell geometry and potentially acts as a signal to trigger mechanosensing pathway. Agrin as a Tissue and ECM Stiffness Signal to Activate YAP We next investigated if ECM stiffness modulated Agrin and its downstream signaling, thereby affecting YAP’s mechanoresponse. Similar to the observations on hydrogels, ECM stiffness enhanced Agrin levels by
2.5- to 4 fold and increased FAK activation and expression of the Agrin co-receptors Lrp4 and MuSK in 3D spheroids over a period of 3–6 days (Figures 3A and 3B). Consistently, depleting Agrin in spheres enclosed by hard, but not compliant, matrices significantly reduced the mRNA levels of YAP target genes (Figure 3C). Conversely, exogenous Agrin increased the expression of these genes in soft ECM (Figure 3D). MIHA cells cultured on soft gels coated with increasing amounts of Agrin displayed decreased YAP phosphorylation and enhanced levels of YAP targets (Axl and Jagged-1) in a doseresponsive manner (Figure 3E). Consistently, Agrin also activated FAK (Figure 3E), suggesting that Agrin induces mechanosignaling to activate YAP. Since our data indicate that ECM stiffness enhances Agrin levels, we next probed whether Agrin enhances ECM/ tissue stiffness. Accordingly, we measured the in vivo collagen content by picrosirius red staining of control and Agrin-depleted tumors as a qualitative index for tissue stiffness (Levental et al., 2009). Intense fibrillar collagen staining in control tumors signifying higher ECM stiffness was strikingly reduced in Agrin-depleted xenografts but was significantly restored by exogenously expressing Agrin in Agrin-depleted tumors (Figure 3F). These results reveal that Agrin-induced YAP activation may lead to collagen accumulation that contributes toward ECM/tissue stiffness. As geometry and substrate rigidity affect cell stiffness (Tee et al., 2011), we subsequently tested whether Agrin, as a stiffness cue, enhances cellular contractility to activate YAP in compliant substrates. To this end, we embedded MIHA cells in low-density collagen gel that recapitulate soft matrices. In these situations, ECM stiffness cues stimulate cell contractility that is qualitatively approximated by the degree of gel shrinkage. Owing to the low endogenous Agrin levels in compliant matrix,

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Figure 2. YAP Mechanotransduction Is Regulated by Agrin (A) Indicated cell lines in hard or soft gels for 3 days were subjected to western blot analysis. b-Actin served as a loading control. (B) Representative confocal images of YAP in control or Agrin knockdown MHCC-LM3 cells grown as sparse or medium dense confluency on hard gels. Scale bar, 10 mm. (C) MHCC-LM3 cells in soft gels were either left untreated or treated with indicated concentrations of neural-Agrin for 1 day and processed for YAP immunofluorescence. Representative confocal images are shown with boxed areas as enlarged sections. Scale bar, 10 mm. (D and E) Control or Agrin-depleted MHCC-LM3 spheres in stiff (D) or soft ECM (E) for 6 days and were processed for YAP immunofluorescence. Representative confocal and bright-field images are shown. Sphere diameters from at least four to six fields were analyzed by ImageJ (n = 3, Student’s t test, **p < 0.005). Boxed regions represent enlarged panels. Arrows indicate YAP localization (E). Scale bar, 10 mm. (F and G) Control, Agrin-depleted, or Agrin-depleted MHCC-LM3 cells treated with 20 mg/mL rAgrin were plated on large square patterns (F, left). Untreated or rAgrin-treated MHCC-LM3 cells were cultured in small patterns (F, right). 1 day later, they were processed for YAP and actin-phalloidin staining. More than 50 cells were analyzed for nuclear/cytoplasmic YAP (G) (n = 4, Student’s t test, **p < 0.005 and *p < 0.05, respectively). See also Figure S2.

untreated MIHA cells failed to contract collagen (Figure 3G). Interestingly, exogenous Agrin stimulated these cells to exert greater contractile force resulting in significant collagen contraction (Figure 3G). Importantly, Agrin also induced actin stress fibers and nuclear YAP localization in these cells during contraction (Figures S3A and S3B). However, Agrin failed to stimulate YAP depleted MIHA cells to contract collagen gels, implicating that YAP is essential for ECM remodeling induced by Agrin (Figure 3H). In contrast, Agrin depleted MHCC-LM3 cells had a significant inhibition in collagen contraction when compared to control cells (Figure 3I). Further, soft collagen gels released from the substratum (floating) are inherently softer compared to the attached ones despite maintaining comparable collagen

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binding site concentrations (Figure 3J). Interestingly, neuralAgrin-treated MIHA cells suspended in floating collagen gels strongly shifted YAP to the nucleus (Figure S3C). This was also accompanied with >3-fold increase in the stiffness of these floating matrices (Figure 3J). Conversely, Agrin-depleted cells conferred less stiffness to the floating matrices (Figure S3D), suggesting that Agrin confers greater contractile strength to cells due to its ability to generate a stiff ECM. Agrin Requires FAK to Maintain YAP Activity in a Stiffness-Sensing Manner Consistent with a role of FAK and Src in regulating YAP activity, Agrin-FAK synergism regulates tumorigenesis (Chakraborty

Figure 3. ECM Stiffness and Agrin Confer Rigidity to Regulate YAP Activity (A and B) MHCC-LM3 spheres as in Figures 2D and 2E for indicated days were analyzed by western blot for Agrin (A) and indicated proteins (B). b-Actin served as a loading control. Agrin levels were normalized to that of b-actin (A). (C) RT-PCR analyses for YAP target genes in spheres from (A) and (B) (n = 3, Student’s t test, *p < 0.05 and **p < 0.005, respectively). (D) RT-PCR analyses in untreated or neural Agrin (10 mg/mL) treated MHCC-LM3 spheres in soft ECM for 6 days (n = 3; data are presented as mean ± SD, *p < 0.05 and **p < 0.005, respectively). (E) MIHA cells cultured in soft gels coated with increasing concentrations of Agrin for 2 days were analyzed by western blot for the indicated proteins.

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et al., 2015; Kim and Gumbiner, 2015). Hence, we examined whether Agrin requires FAK in regulating YAP in the context of matrix stiffness. Compared to the control, FAK inhibition by PF-562271 reduced sphere growth in stiff ECM (Figure S4A). While cells of control spheres had nuclear YAP distribution, PF-562271 treatment resulted in smaller spheres with cytosolic YAP after 10 days in stiff ECM (Figure S4B). Supplementing Agrin to spheres enclosed by soft ECM stimulated their growth that was significantly suppressed by FAK inhibition (Figure S4C). Agrin also shifted YAP into the nucleus and induced gel shrinkage, even in spheres enclosed in soft ECM (Figures S4D and S4E). Agrin-induced nuclear localization of YAP and gel contraction were also inhibited by PF-562271 (Figures S4D and S4E). As FAK inhibition consistently enhanced YAP phosphorylation (Figures S1A and S4F), these data support that Agrin requires FAK to mediate cell contractility to sustain YAP activation and liver cancer cell sphere growth. Agrin-FAK Signaling Inactivates the Hippo Pathway To gain mechanistic insights into the role of Agrin and FAK in regulating YAP and growth in response to matrix stiffness, we analyzed the activity of the Hippo components in Agrin- or FAK-depleted cells. Interestingly, Amot-p130 phosphorylation mediated by the core Hippo kinases (Adler et al., 2013; Chan et al., 2013) was greatly enhanced with Agrin or FAK depletion (Figure 4A). Consistently, Mst1/2 and LATS1/2 were also activated by Agrin or FAK knockdown (Figure 4A). The total protein levels of the core Hippo components remained unchanged (Figure 4A). Similar effects were also observed in MCF-7 cells (Figure 4A). The mammalian Hippo pathway is activated by Merlin/NF2, which leads to activation of Mst1/2 and LATS1/2 kinases (Hamaratoglu et al., 2006; Hong and Guan, 2012; Zhang et al., 2010). Merlin activity is tightly controlled by phosphorylation at Ser518, which, in turn, inactivates its tumor-suppressive function (Jin et al., 2006; Li et al., 2010). Recently, ILK was shown to modulate Merlin and inactivate the Hippo pathway (Serrano et al., 2013). Interestingly, Merlin was constitutively phosphorylated at Ser518 residue in liver cancer cells (thus present in an inactive form) (Figure 4A). Agrin or FAK knockdown decreased Merlin phosphorylation (and thus activated Merlin) along-with significant reductions in ILK levels (Figure 4A), suggesting that Agrin-FAK signaling inactivates the core Hippo components by maintaining Merlin in an inactive status and sustaining the levels of ILK. To evaluate how Agrin negates the Hippo core components, we considered the fact that Merlin is phosphorylated at Ser518

by PAK1 downstream of integrin-Rac signaling (Kissil et al., 2003; Okada et al., 2007). As Agrin knockdown decreased Merlin Ser518 phosphorylation, we next tested whether Agrin facilitates the PAK1-Merlin interaction. Like Rac1 (Okada et al., 2007), we found that Merlin and, surprisingly, ILK, are interacting partners enriched with PAK1-PBD (Figure 4B, left). Agrin depletion severely compromised the formation of this ILK-Rac-PAK1Merlin complex (Figure 4B, left). Stable knockdown of Agrin in Hep3B cells also showed a similar loss of interaction of ILK and Merlin with PAK1-PBD (Figure 4B, right). Importantly, supplementing recombinant Agrin (rAgrin) in knockdown cells substantially restored these interactions with PAK1-PBD, suggesting that Agrin facilitates the formation of the ILK-PAK1Merlin protein complex, likely serving to inactivate Merlin (Figure 4B, left panel). Depletion of FAK also decreased this association, albeit to a lesser extent when compared to Agrindepleted cells (Figure 4B, left). To confirm whether Agrin inactivates Merlin through the PAK1-ILK pathway, we utilized MIHA cells that have low endogenous Merlin phosphorylation under normal culture conditions (Figure 4C). Supplementing neural Agrin stimulated Merlin phosphorylation and increased ILK protein levels in a dose-responsive manner (Figure 4C). Moreover, treatment of these cells with IPA3 (a selective PAK1 inhibitor) largely suppressed this Agrin-induced phosphorylation of Merlin (Figure 4D). Consistently, ECM stiffness increased Merlin and PAK1 phosphorylation together with higher ILK protein levels in MHCC-LM3 spheres (Figure 4E). Therefore, correlating with the fact that Agrin signaling was enhanced with ECM stiffness, these results reveal that ILK-PAK1 mediates inhibitory Merlin phosphorylation in response to exogenous Agrin and stiff ECM. Agrin Restricts Focal Adhesome Enrichment of Hippo Components To further determine if Agrin alters the composition of FAs to regulate the Hippo components at the interface of cell-matrix adhesions, FAs from control and Agrin-depleted cells were isolated (Kuo et al., 2011) (Figure 4F). The ‘‘focal adhesome’’ was enriched in FAK, paxillin, and vinculin proteins, whereas the ‘‘cell body’’ was enriched in actin, GAPDH, and Rho-GDI (Figure 4F). Agrin knockdown did not affect the levels of bona fide FA proteins (Figure 4F). Interestingly, the Hippo components, including Amot, Mst1/2, LATS1/2, and Merlin, were minimally associated with focal adhesomes of control cells; however, Agrin depletion significantly enriched them in the adhesomes (Figure 4G). In addition to the core Hippo components, the b-Pix level also increased within FAs upon Agrin knockdown (Figure 4G). b-Pix reportedly activates Hippo signaling by interacting with

(F) Sectioned Control, Agrin-depleted, or rescued tumors were stained with picrosirius red. Fibrillar collagen content levels are shown (n = 3 mice per group, four to six sections analyzed). Scale bar, 100 mm. (G) MIHA cells in soft ECM were either treated with neural Agrin (10 mg/mL) or left untreated. After 1 day, the gel was released and relative contraction measured at indicated time (n = 4, Student’s t test, **p < 0.005). (H) Control or YAP-depleted MIHA cells treated with neural Agrin (10 mg/mL) were subjected to gel contraction assay as in (G). Relative gel shrinkage is quantified as in (G) (n = 3, triplicates, Student’s t test, *p < 0.05). (I) Control or Agrin-depleted MHCC-LM3 cells were subjected to gel contraction assay and represented as in (G) (n = 2, triplicates, Student’s t test, *p < 0.05 and **p < 0.005, respectively). (J) Untreated or 5 mg/mL neural-Agrin-treated MIHA cells were suspended on soft floating collagen gels for 2 days. Matrix stiffness was measured by AFM and is represented as relative fold change (n = 3, Student’s t test). See Figures S2 and S3.

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LATS1/2 and YAP and negatively regulates FAs (Heidary Arash et al., 2014; Kuo et al., 2011). In contrast, PAK1 and ILK, the negative regulators of the Hippo pathway, were significantly reduced in FAs upon Agrin depletion (Figure 4G). As early as 10 min post-adhesion on stiff gels, b-Pix was excluded from the activated FAs marked by pFAK (Tyr397) in control cells (Figure 4H, i). Agrin-depleted cells exhibited increased colocalization between pFAK and b-Pix at cell-matrix adhesions, which was significantly reduced in knockdown cells supplemented with neural Agrin (Figure 4H, ii and iii). Conversely, substantial colocalization between pFAK and b-Pix observed in cells adhered to soft gels was largely inhibited by exogenous Agrin (Figure 4H, iv and v). Cumulatively, these results suggest that Agrin restricts the assembly of b-Pix and Hippo signaling in FAs.

LATS1/2 in Agrin knockdown cells. As expected, increased YAP phosphorylation upon Agrin depletion was suppressed by Merlin or LATS1/2 knockdown (Figure 5D). In the context of ECM stiffness, LATS1/2 depletion in spheres grown in hard ECM increased Cyr61 and CTGF mRNA levels (Figure 5E, left). Agrin knockdown in such a setting reduced YAP target mRNA levels by
81 and 84%, respectively (Figure 5E, right). Consistently, this inhibition of CTGF and Cyr61 mRNA expression was significantly restored by depleting LATS1/2 in Agrin knockdown cells (Figure 5E, right). Collectively, these results reveal that (1) Agrin plays opposing roles with Merlin and LATS1/2 and (2) inactivation of YAP in Agrin-depleted cells is mainly mediated by Merlin and LATS1/2, which correlates with the notion that Agrin suppresses Merlin and LATS1/2 to activate YAP.

Agrin Negates the Tumor-Suppressive Roles of Merlin and LATS1/2 By engaging ILK-PAK1 signaling in Merlin-expressing cells, it is possible that Agrin overcomes the tumor suppression of Merlin and LATS1/2 to exert its oncogenic functions. To substantiate this, we first examined if Merlin and LATS1/2 were growth suppressive in liver cancer cells. Expressing Merlin in MHCC-LM3 cells increased YAP phosphorylation and significantly reduced migration as compared to vector-transfected cells (Figures S5A and S5B). Conversely, depleting Merlin in these cells reduced LATS1/2 and YAP phosphorylation; LATS1/2 knockdown also inhibited YAP phosphorylation (Figures S5C and S5D). Similarly, LATS2 expression increased YAP phosphorylation and decreased cellular migration (Figures S5E and S5F). Thus, having established the tumor suppressive roles of Merlin and LATS1/2, we tested the interplay of opposing functions of Agrin and Merlin. Owing to low endogenous expression of both Merlin and Agrin, we expressed Merlin-hemagglutinin (MerlinHA) and Agrin-GFP in MIHA cells (Figure 5A). Compared to cells transfected with vector or Merlin-HA, Agrin-GFP substantially reduced YAP phosphorylation (Figure 5A). However, when Agrin and Merlin were co-expressed, YAP phosphorylation remained high (Figure 5A). Similarly, Agrin-induced cell migration was inhibited by co-expressing Merlin (Figure 5B), therefore supporting the notion that Merlin, acting downstream of Agrin, may be functionally antagonistic. Additionally, the interaction between Merlin and LATS1 was dramatically enhanced upon Agrin knockdown (Figure 5C). To further assess whether Merlin and LATS1/2 inactivated YAP upon Agrin knockdown, we depleted Merlin and

Agrin Regulates the Mechanoactivity of YAP through Cytoskeletal Modulation in Merlin Null Cells The sensitivity of YAP to mechanical cues in Merlin-deficient MDA-MB-231 cells has often challenged the role of Hippo signaling in mechanotransduction (Dupont et al., 2011). Despite negating the functions of Merlin and LATS1/2, we further investigated whether Agrin also regulates YAP in Merlin null cells, most likely without engaging an ILK-PAK1 signaling. Indeed, increased YAP phosphorylation and reduced proliferation were evident in Agrin-depleted MDA-MB-231 cells (Figure 5F). Moreover, in hard ECM, nuclear YAP of control cells was shifted to the cytoplasm upon Agrin knockdown (Figure 5G). As observed in liver cancer cells, Ser127 phosphorylation and cytoplasmic YAP in compliant substrates was strongly reduced by exogenous Agrin in MDA-MB-231 cells, thereby activating YAP in the nucleus (Figures 5H and 5I). Importantly, this Agrin-induced de-phosphorylation, nuclear YAP localization, and expression of its target genes Jagged-1 and Cyr61 were blocked by RhoA and ROCK inhibitors (Clostridium botulinum exoenzyme C3 and Y27632, respectively) and F-actin blocking agent (latrunculin B) (Figures 5H and 5I). Agrin is known to induce actin polymerization and cytoskeletal reorganization, and actin stress fibers collaborate with the Hippo pathway (Cartaud et al., 2011; Chakraborty et al., 2015; Wada et al., 2011). Therefore, not surprisingly, these inhibitors blocked Agrin-induced nuclear YAP localization and shifted YAP to the cytoplasm even in MHCC-LM3 cells (Figure 5H). These data suggest an additional layer of YAP regulation through the RhoA-actin cytoskeleton by Agrin, which is more prominent in Merlin-deficient cells.

Figure 4. Inactivation of the Hippo Pathway by Agrin-FAK-ILK Signaling (A) Control, Agrin-, and FAK-depleted cells were analyzed by western blots for indicated proteins. b-Actin served as a loading control. (B) MHCC-LM3 cell lysates from control, Agrin and FAK knockdown, and Agrin-depleted cells rescued by neural Agrin (10 mg/mL) for 1 day were precipitated with PAK-PBD beads and analyzed by western blot. 10% total lysates were used as input controls. PAK-PBD beads were detected by Coomassie brilliant blue (CBB) stain. Agrin-depleted Hep3B cells were used for same experiment (right). (C) MIHA cells treated with increasing concentrations of neural Agrin for 1 day were analyzed by western blot for indicated proteins. b-Actin served as a loading control. (D) MIHA cells were pre-treated with solvent or 10 mM IPA3 for 4 hr in the presence or absence of 10 mg/mL rAgrin for 12 hr and analyzed by western blot for indicated proteins. (E) Western blot analysis in MHCC-LM3 cells in hard and soft ECM for 6 days. (F and G) Western blot analysis in control and Agrin-depleted MHCC-LM3 cells for FA and cell body markers (F), respectively, and other indicated proteins (G). (H) Control, Agrin-depleted, and Agrin-rescued MHCC-LM3 cells were plated on stiff gels (i–iii) or untreated and Agrin-treated cells in soft gels (iv and v) were processed for pFAK and b-pix staining at 10 min post-adhesion. Representative confocal images are shown. Scale bar, 10 mm. See Figure S4.

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Figure 5. Agrin Negates Merlin-LATS and Modulates the RhoA-Dependent Actin Cytoskeleton in Merlin-Deficient Cells (A and B) MIHA cells expressing vector, Merlin-HA, Agrin-GFP, and Agrin-GFP and Merlin-HA together were subjected to western blot analysis for indicated proteins (A) or migration assay (B) after 3 days. For (B), migrating cells were imaged (103 magnification) and counted by ImageJ (n = 3, mean ± SD, **p < 0.005). (C) Immunoprecipitation with Merlin antibody in control and Agrin-depleted MHCC-LM3 cell lysates for detecting LATS1. 10% protein lysates were used as input. (D) MHCC-LM3 cells were transfected with control or Agrin siRNA alone or in combination with either Merlin (left) or LATS1/2 siRNAs (right). Western blot was done at day 3 to detect the indicated proteins. (E) RT-PCR analysis of indicated genes in control and LATS1/2 knockdown MHCC-LM3 spheres in stiff ECM for 3 days (left). RT-PCR for same genes in control, Agrin-depleted, or Agrin and LATS1/2 co-depleted spheres in stiff ECM, respectively (right) (n = 3, Student’s t test, *p < 0.05 and **p < 0.005, respectively). (F) Western blots for the indicated proteins in control and Agrin-depleted MDA-MB-231 cells. Relative absorbance (490 nm) from MTS assay normalized to control is shown (n = 4, Student’s t test, *p < 0.05). (G) Confocal images of control and Agrin-depleted MDA-MB-231 cells showing YAP and actin staining. Cytoplasmic/nuclear YAP localization was quantified in 150–200 cells. Scale bar, 10 mm. (H and I) Indicated cells in soft gels were either untreated or treated with rAgrin (10 mg/mL) for 1 day in the absence or presence of indicated inhibitors for 1 hr, and processed for immunofluorescence to detect YAP (H). Cytoplasmic/nuclear staining of YAP quantified as in (G). Scale bar, 10 mm. MDA-MB-231 cells were analyzed by western blot analysis (I) for the indicated proteins. See Figure S5.

Integrin-Lrp4/MuSK Signaling as Important Scaffolds for Agrin in the Regulation of YAP Binding of Agrin to Lrp4/MuSK is critical for the oncogenesis of HCC through the activation of integrin b1-FAK pathway (Chakraborty et al., 2015). While Agrin depletion reduced the phosphorylation of MuSK in cancer cells, neural Agrin treatment, in contrast, stimulated dose-dependent MuSK phosphorylation in MIHA cells, suggesting that Agrin induces MuSK phosphoryla-

tion in liver cells (Figure 6A). Therefore, to determine whether ECM rigidity and Agrin signals are relayed through the Lrp4/MuSK and integrin pathways, we hypothesized that cumulative inhibition of Agrin and its downstream signaling may completely inactivate YAP, while perturbing either the Lrp4/MuSK pathway or the integrin pathway may inhibit YAP in a less robust fashion. Though Lrp4 and MuSK depletion displayed enhanced YAP phosphorylation (Figures 6B and S6A,

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Figure 6. ECM Stiffness Is Relayed by the Agrin-MuSK-Integrin Pathway to YAP (A) Detection of MuSK phosphorylation in control or Agrin-depleted MHCC-LM3 cells (left). MIHA cells treated with or without indicated concentrations of neural Agrin were analyzed as above for MuSK phosphorylation (right). (B) Western blot analysis for indicated proteins in MHCC-LM3 cells transfected with either the indicated siRNAs alone or in combination with Agrin siRNA. (C) Western blot analysis in Control or Agrin depleted MHCC-LM3 cells expressing vector or rat-MuSK-GFP. b-actin served as a loading control. (D) Control or Agrin-depleted MHCC-LM3 cells either untreated or treated with 500 mg/mL RGD for 1 day were analyzed by western blot for indicated proteins. (E and F) Western blot (E) and RT-PCR (F) analysis in MHCC-LM3 spheres in soft matrix treated with 20 mg/mL fibronectin or Agrin for 1 day (n = 3, Student’s t test, **p < 0.005 and *p < 0.05, respectively). (G) Western blot analysis in MHCC-LM3 cells transfected with the indicated siRNAs alone or along-with 500 mg/mL RGD.

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first two panels), this effect was amplified when Agrin was simultaneously depleted in MuSK or Lrp4 knockdown cells (Figure 6B). Moreover, when compared to mild differences in control cells, overexpression of MuSK in Agrin knockdown cells substantially reduced YAP phosphorylation (Figure 6C), suggesting that Lrp4-MuSK mediates part of Agrin function. The role of dystroglycan (DG), another Agrin receptor, was ruled out, as its depletion did not affect YAP (Figure S6A, third panel). Owing to their ability to communicate with and assess ECM mechanics (Burkin et al., 2000; Chakraborty et al., 2015; Humphrey et al., 2014; Martin and Sanes, 1997; Ross et al., 2013; Schiller et al., 2013), we directly tested the role of integrins on YAP phosphorylation in conjunction with Agrin-Lrp4/MuSK. Although RGD peptides (which inhibit integrin signaling) reduced cell migration and FAK activity (Figures S6B and S6C, second panels) and robustly increased YAP phosphorylation (Figure 6D), these effects were more striking when RGD peptides were treated in Agrin-depleted cells (Figures S6B, S6C, and 6D). As such, knockdown of integrin b1 increased YAP phosphorylation in part, but the resulting effect in Agrin and integrin doubledepleted cells was consistently higher and comparable to that of Agrin-depleted cells (Figures S6D and S6E). Inactivation of ILK and PAK1 also had effects similar to integrin inhibition (Figures S6F–S6H). Integrin depletion in cells on stiff ECM also resulted in a substantial shift of nuclear YAP into the cytoplasm (Figure S6I). Consistently, depleting Agrin in integrin knockdown cells shifted YAP more robustly into the cytoplasm, once again suggesting a scenario of enhanced YAP inactivation (Figure S6I). Therefore, these results indicate that upon knockdown of integrins, Agrin partly utilizes a Lrp4/MuSK scaffold; however, depletion of Agrin completely perturbed signal transduction through both signaling pathways contributing to maximal YAP inactivation. However, integrin stimulation alone was not sufficient to activate YAP in soft matrices. While both fibronectin and Agrin treatment activated FAK in compliant substrates, only exogenous Agrin considerably decreased YAP phosphorylation and increased YAP target mRNA levels (Figures 6E and 6F). Stimulation of integrins by fibronectin was not sufficient to alter YAP phosphorylation and its target genes when endogenous Agrin levels were low in compliant ECM (Figures 6E and 6F). These lines of evidence indicate that Agrin is an indispensable ECM signal recognized by the integrin-Lrp4/MuSK complex coordinating ECM stiffness to YAP mechanosensing. Consistently, blocking MuSK and integrins simultaneously inactivated YAP in a manner similar to that observed upon Agrin depletion. We observed robust YAP phosphorylation when MuSK-depleted cells were treated with RGD when compared to RGD treatment or MuSK depletion alone (Figure 6G). In fact, the YAP phosphorylation levels in MuSK-depleted cells treated with RGD were identical to those of Agrin-depleted cells inactivated for integrin signaling (Figure 6G), confirming that MuSK and integrins collectively are critical downstream partners for

Agrin to activate YAP. In addition, compared to untreated control cells in stiff ECM, RGD and MuSK small interfering RNA (siRNA) partly perturbed nuclear YAP distribution (Figure 6H, i–iii). Interestingly, RGD treatment in MuSK-depleted cells essentially shifted YAP into the cytoplasm (Figure 6H, iv). This observation mirrored that of Agrin knockdown cells on stiff substrates (Figure 6H, v). Although MuSK depletion or RGD treatment each partly reduced the expression of Jagged-1 and Cyr61, the combined inhibition of MuSK and integrin pathways drastically inhibited their expression (Figure 6I). Hence, inhibiting both MuSK and integrin signaling pathways downstream of Agrin disrupts YAP’s mechanoresponsiveness to ECM stiffness. Agrin-Mediated Oncogenesis Is Dependent on YAP Finally, to address the relevance of Agrin’s mechanotransducing effects on YAP, we depleted YAP in HCC cells (Figure 7A). Cell proliferation and migration induced by exogenous Agrin in HCC cells was significantly reduced by YAP depletion (Figures 7A and 7B). Furthermore, Agrin stimulated anchorage-independent growth in soft agar, and enhanced CTGF and Cyr61 mRNA was also suppressed by YAP depletion (Figures 7C and 7D). As expected, YAP knockdown also reduced these oncogenic properties (Figures 7A–7D). As demonstrated previously, Agrin knockdown severely inhibited tumor growth that was significantly rescued by expressing rat-Agrin construct (Figure 7E) (Chakraborty et al., 2015). To decipher whether Agrin requires transcriptional activity of YAP for tumorigenesis, we tested the effects of the YAP-TEAD inhibitor verteporfin (VP) on the tumor growth of Agrin-rescued cells. Agrin-induced tumor growth and YAP target gene (AXL and Cyr61) expression were significantly suppressed by VP treatment (Figures 7E and 7F). In a complementary approach, we rationalized whether YAP activation rescued the oncogenic defects in Agrin knockdown cells. Hence, rat Agrin, wild-type YAP, or YAP-5SA (constitutively active) was expressed in Agrin knockdown cells (Figure 7G). Compared to the inhibition observed in parental or vector-transduced Agrin-depleted cells, a robust increase in the YAP target protein Jagged-1 was observed upon rat Agrin, YAP, or YAP-5SA expression (Figure 7G). Accordingly, reduced cell proliferation in Agrin knockdown cells was rescued by YAP or YAP5SA expression in a manner comparable to that of Agrin-rescued cells (Figure 7G). Strong nuclear accumulation of YAP was observed in wild-type or YAP5SA expressing Agrin knockdown cells (Figure 7H). Similar to Agrin rescue, YAP or YAP5SA expression restored cell migratory and anchorage-independent growth defects of Agrindepleted cells (Figures 7I and 7J), thereby establishing YAP as a potent downstream effector for Agrin-mediated oncogenesis. Compared to low levels of Agrin and cytosolic YAP distribution in normal liver tissues, increased Agrin expression and more nuclear YAP enrichment were observed in 81% of HCC tissues (Figure S7A). Higher Agrin mRNA correlated with poor survival among liver cancer patients (Figure 7K). Consistent with our findings thus far, elevated gene expression of Agrin and YAP targets

(H) Control or MuSK-depleted MHCC-LM3 cells in stiff gels were treated with 500 mg/mL RGD for 1 day to detect YAP and actin immunofluorescence. Agrin-depleted cells were used as controls. Scale bar, 10 mm. (I) Cell lysates treated as in (H) were analyzed by western blot for the indicated proteins. See also Figure S6.

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Figure 7. Agrin Depends on YAP for Its Oncogenic Activities (A) Western blot for YAP knockdown in the indicated cell lines. MTS assay was done in control and shYAP#2 cells either untreated or treated with rAgrin (20 mg/mL) on the indicated days (n = 3, Student’s t test, **p < 0.005). (B and C) MHCC-LM3 cells treated as in (A) were subjected to migration (B) and soft agar assay (C). Migrating cells (B) were imaged at 103 magnification and quantified using ImageJ (n = 3, mean ± SD, ***p < 0.0005 and **p < 0.005, respectively). Soft agar colonies (C) after 14 days were quantified using ImageJ (n = 3, Student’s t test, ***p < 0.0005). (D) RT-PCR analysis in cells from (B) for CTGF and Cyr61 (n = 3; data represent mean ± SD, Student’s t test, **p < 0.005 and *p < 0.05).

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(CYR61, IGFBP3, AMOTL, and AXL) together resulted in even poorer survival in patients (Figure S7B). Cumulatively, these results support that Agrin engages YAP-mediated transcription to mediate oncogenesis. DISCUSSION Here, we identified a unique role of Agrin in regulating the Hippo pathway at the cell-matrix interface. Several major conclusions advanced our understanding of Agrin, the Hippo pathway, and mechanotransduction related to cancer development. First, as Agrin and its signaling cascade to YAP are upregulated in rigid ECM, it may thereby serve as a tissue rigidity signal. Second, although we previously revealed a role of Agrin in HCC (Chakraborty et al., 2015), its link to the Hippo pathway and YAP regulation was unknown. We hereby suggest that YAP activation in response to extracellular Agrin is important for rigidity-mediated signaling and essential for the oncogenic property of Agrin. Third, Agrin expression correlates with the clinical progression of HCC. The combined correlation between Agrin and YAP target genes and poorer survival offers clinical relevance to our discovery. Fourth, in Merlin-expressing cells, Agrin may sustain PAK1 to mediate phosphorylation of Merlin and suppress its tumorsuppressor function. Clearly, Agrin engages both integrins as well as Lrp4/MuSK for optimal activation of YAP in response to ECM stiffness. Interestingly, as shown in Merlin-deficient cells, Agrin also remains capable of relaying signals to YAP through RhoA-dependent actin polymerization. Given that tissue stiffness correlates with tumor malignancy (DuFort et al., 2011), the Agrin-mediated mechanotransduction may play a more general role within the tumor microenvironment. Mechanistically, we propose a model where ECM rigidity induces Agrin to act as a cellular contractility and stiffness signal relayed to YAP via combinatory activation of Lrp4/MuSK and integrin pathways (Figure S7C). In this context, although MuSK, in part, is important in relaying Agrin’s signal to YAP, our results also suggest that Agrin may engage alternate pathway(s), including integrin and actin polymerization bypassing MuSK in some situations. Our results are consistent with the notion that activation of integrins by Agrin is critical for the YAP-mechanotransduction pathway. This, in turn, activates FAK, ILK, and PAK1 as an important scaffold. Agrin antagonizes the Hippo signaling by minimizing their enrichment within focal adhesomes. In stiff ECM, Agrin stimulates ILK-PAK1 to inactivate Merlin, which outlines an opposing interplay between Agrin with Merlin and LATS1/2. Although we and others have empha-

sized that FAK is required for YAP activity (Kim and Gumbiner, 2015), our results also suggest that integrin and FAK activity are insufficient to activate YAP when Agrin levels were low or depleted in compliant substrates and in fibronectin patterns. While the YAP-mechanotransduction may or may not depend on LATS1/2 based on different contexts (Low et al., 2014), our results show that inactivation of YAP in stiff ECM upon Agrin depletion requires functional Merlin and LATS1/2. Importantly, the incapability of LATS1/2 or Merlin knockdown to rescue YAP activity in compliant matrices (Dupont et al., 2011) may also be partly attributed to the low levels of Agrin in such situations. In summary, these findings suggest that Agrin likely serves as a mechanotransduction signal for YAP, therefore underlining liver cancer development. EXPERIMENTAL PROCEDURES Cell Lines The HCC cell line Hep3B 2.1 was purchased from American Type Culture Collection (ATCC) and cultured using the recommended media. All other cell lines were cultured as before (Chakraborty et al., 2015). Generation of Knockdown Cells Agrin knockdown was performed by lentiviral shRNAs against human Agrin (Chakraborty et al., 2015). Agrin shRNA sequences were 50 -CAGGAGAA UGUCUUCAAGATT-30 , 50 -CGACGUGUGCUGUGAAGAATT-30 , 50 -CGACCU CUUCCGGAAUUCATT-30 ; YAP shRNA sequences were 50 -GCCACCAAGC TAGATAAAGAA-30 and 5-CCCAGTTAAAATGTTCACCAAT-30 ; siRNAs were Agrin (L-031716-00-0050), Lrp4 (L-027194-02-0020), FAK (L003164-0020), integrin b1 (L-004506-010), ILK (L-004499-0020), LATS1/2 (L-004692-005 and L-003865-005), MuSK (L-003158-010), and control (D-001210-01-20) were from Dharmacon (Thermo Fisher Scientific); and MuSK (AM16704), Merlin (AM16704), and PAK1 (AM16708) siRNAs were from Ambion Life Technologies, and all transfections were performed as before (Chakraborty et al., 2015). In Vivo Tumorigenesis 4- to 6-week-old female nude mice were subcutaneously (s.c.) injected with 1 3 107 MHCC-LM3 cells/mL suspended in Matrigel. Once the tumors reached a size of
200 mm3, the mice were either treated with four doses of PBS control or Agrin antibody (10 mg/kg every 4 days). Mice were sacrificed on day 38. For VP treatment, nude mice were similarly injected with Agrin knockdown MHCC-LM3 cells alone or those rescued with rat Agrin. When the tumors reached 70 mm3, the mice bearing Agrin-rescued cells were divided into two groups and were treated with vehicle or VP (100 mg/kg) daily for 20 days. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC; 161111), Agency for Science, Technology and Research (A*STAR), Singapore. Statistical Analysis All experiments were performed at least three times. Data represent mean ± SD from triplicate experiments. For in vivo experiments, data are presented as mean ± SEM. A paired two-tailed Student’s t test was performed

(E) Western blots showing Agrin and actin expression in knockdown cells rescued by rat Agrin. Tumor photograph and mean tumor volume derived from parental Agrin knockdown cells with vector (n = 5) or rescued cells treated with either vehicle or VP are shown (n = 6; data represent mean ± SEM, Student’s t test, **p < 0.005 and ***p < 0.0005). (F) RT-PCR analysis in tumors from (E) detecting YAP target genes normalized to 18S rRNA. Data are presented as mean ± SD (n = 3, Student’s t test, **p < 0.005). (G and H) Control, Agrin-depleted, and knockdown MHCC-LM3 cells expressing vector, rat Agrin, wild-type, and YAP5SA constructs were analyzed by western blot after 2 days (G). MTS assay was done in same cells after 3 days (G). Relative absorbance normalized to control shRNA cells are shown (n = 4, Student’s t test, **p < 0.005). The same cells were analyzed by immunofluorescence for YAP localization (H). Cytoplasmic/nuclear localization of YAP was quantified in 150–200 cells. Scale bar, 10 mm. (I and J) Cells from (G) and (H) were subjected to a migration assay (I) or soft agar assay (J) and presented as in (B) and (C) (n = 3, Student’s t test, **p < 0.005). (K) Survival curves in The Cancer Genome Atlas (TCGA) liver cancer patients grouped based on high (red) or low (green) Agrin expression (Student’s t test, p < 0.00016). See also Figure S7.

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using GraphPad Prism. Data were considered significant when *p < 0.05; **p < 0.005; and ***p < 0.0005, respectively. Other experimental procedures are described in Supplemental Experimental Procedures.

Chan, S.W., Lim, C.J., Guo, F., Tan, I., Leung, T., and Hong, W. (2013). Actinbinding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation. J. Biol. Chem. 288, 37296–37307.

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AUTHOR CONTRIBUTIONS S.C. and W.H. conceived the project. S.C. designed and performed all the experiments with assistance from K.N. and A.V.P. Y.B.L., S.C., and C.T.L. performed atomic force microscopy (AFM) experiments. S.C., A.R., and M.L. performed the in vivo experiments with input from V.T. S.C. analyzed the data and wrote the manuscript with input from W.H. ACKNOWLEDGMENTS The study was funded by Early Career Research (ECR), Institute of Molecular and Cell Biology (IMCB) (S.C.) and Agency for Science, Technology and Research (A-STAR) funds (W.H.). We thank Dr. Steven J. Burden (NYU School of Medicine) for myc-MuSK-GFP. We acknowledge SiewWee Chan and Chenying Liu for providing reagents. We thank the Advanced Molecular Pathology laboratory at IMCB for assistance with immunohistochemistry. Received: April 19, 2016 Revised: December 28, 2016 Accepted: February 13, 2017 Published: March 7, 2017 REFERENCES Adler, J.J., Johnson, D.E., Heller, B.L., Bringman, L.R., Ranahan, W.P., Conwell, M.D., Sun, Y., Hudmon, A., and Wells, C.D. (2013). Serum deprivation inhibits the transcriptional co-activator YAP and cell growth via phosphorylation of the 130-kDa isoform of Angiomotin by the LATS1/2 protein kinases. Proc. Natl. Acad. Sci. USA 110, 17368–17373. Aragona, M., Panciera, T., Manfrin, A., Giulitti, S., Michielin, F., Elvassore, N., Dupont, S., and Piccolo, S. (2013). A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154, 1047–1059.

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