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. 2013 Jun;144(7):1530-1542.e12.
doi: 10.1053/j.gastro.2013.02.009. Epub 2013 Feb 16.

Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma

Darjus Felix Tschaharganeh  1 , Xin Chen, Philipp Latzko, Mona Malz, Matthias Martin Gaida, Klaus Felix, Sara Ladu, Stephan Singer, Federico Pinna, Norbert Gretz, Carsten Sticht, Maria Lauda Tomasi, Salvatore Delogu, Matthias Evert, Biao Fan, Silvia Ribback, Lijie Jiang, Stefania Brozzetti, Frank Bergmann, Frank Dombrowski, Peter Schirmacher, Diego Francesco Calvisi, Kai Breuhahn
Affiliations

Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma

Darjus Felix Tschaharganeh et al. Gastroenterology. 2013 Jun.

Abstract

Background & aims: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear.

Methods: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients.

Results: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer.

Conclusions: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

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Conflict of interest statement

Conflict of Interest No conflicts of financial interest to declare.

Figures

Figure 1
Figure 1. YAP-dependent expression of Jag-1 in HCC cells
(A) Comparative intersection analysis of elevated transcripts in murine hepatocytes expressing YAP and HuH-7 cells after YAP silencing (transfection of si#1) revealed 15 inversely regulated candidate genes. Selected candidates were validated in HuH-7 by real-time PCR using an additional YAP-specific siRNA (si#2). (B) Western blot analysis of YAP, Jag-1, and NICD, 24 h after siRNA-mediated knockdown of YAP in HuH-7 cells. (C) Relative mRNA levels of the Notch pathway target gene Hes-1 in HuH-7 cells 24 h after transfection of YAP-specific siRNAs. Untreated cells (cont.) were used for calibration. (D) Western blot analysis of PLC/PRF/5 protein extracts after vector-based overexpression of YAPwt. Amounts of Jag-1, YAPwt, and Hes-1 were detected 24 h after transfection. Untreated (cont.) and empty vector (mock)-transfected cells served as controls. For (B) and (D) actin was used as loading control.
Figure 2
Figure 2. YAP overexpression induces Jag-1/Notch in murine hepatocytes
(A) Transcript levels of YAPS127A, Jag-1, and Hes-1 in primary hepatocytes of LAP-tTA/YAPS127 animals with and without doxycycline treatment 24 h after isolation. For each analyzed gene, the ratio between dox+/dox− is depicted. Untreated hepatocytes were used for calibration. Arrow indicates time-point of doxycycline depletion. (B) Western blot analysis of liver extracts from LAP-tTA/YAPS127A animals fed with (controls, n=3) and without doxycycline (n=4) for 4 weeks. YAP, Jag-1, and Hes-1 protein amounts were detected. Actin was used as loading control. (C) H/E overview staining and representative immunohistochemical analysis for YAP, Jag-1, Notch1, CK19, and Ki-67 in livers from LAP-tTA/YAPS127A mice fed with (control) and without doxycycline for 8 weeks. Arrows indicate densely packed, proliferating hepatocytes. Original magnification: x200 in control livers; ×200 and ×40 in LAP-tTA/YAPS127A mice.
Figure 3
Figure 3. Jag-1 mediates tumor-supporting propertied of YAP in HCC cells
(A) Jag-1 and YAP expression and cell proliferation of PLC/PRF/5 cells after overexpression of Jag-1 and concomitant inhibition of YAP by gene-specific siRNA. Statistics compares Jag-1/scramble siRNA with Jag-1/YAP siRNA. (B) Jag-1, YAP, Hes-1 protein levels and proliferation of PLC/PRF/5 cells after overexpression YAPS127A and simultaneous Jag-1 silencing. Statistical testing compares Jag-1 siRNA and YAPS127A/Jag-1 siRNA. (C) Hes-1 expression and cell proliferation of PLC/PRF/5 cells after expression of YAP and concomitant administration of the Notch transcriptional complex inhibitor SAHM1. Statistics compared SAHM1 with YAP/SAHM1. For (A) and (B) untreated (control) and scramble siRNA-transfected cells served as controls. For (C) untreated and DMSO-treated cells were used as controls. For (A), (B), and (C), cell proliferation was measured 24, 48, and 72 h after transfection. Actin was used as loading control. Values represent means ± SD. Statistical test: Mann-Whitney U. n.s. - not significant, **p<0.01, ***p<0.001.
Figure 4
Figure 4. YAP regulates Jag-1 in a TEAD4-dependent manner
(A) Western blot analysis of Jag-1, YAP, and TEAD4 in HuH-7 cells after siRNA-mediated inhibition of YAP and TEAD4 using two independent siRNAs. (B) Protein analysis of Jag-1, YAP, TEAD1/4, and Hes-1 in PLC/PRF/5 cell lysates after overexpression of YAPwt and concomitant silencing of TEAD1 or TEAD4 by RNAi. (C) Western blot of Jag-1, YAP, myc-tagged YAP, TEAD4, and Hes-1 in PLC/PRF/5 cell lysates after overexpression of TEAD4 and simultaneous expression of dominant negative YAP5SAΔC. Note that YAP is myc-tagged in order to discriminate between exogenous and endogenous protein. (D) Luciferase reporter gene assay testing YAP and TEAD4 responsiveness on three regions of the human Jag-1 gene (region 1–3). Expression of YAP or TEAD4 increased luciferase activity of the region 1 construct, which is partly abrogated after administration of a chemical compound disrupting YAP/TEAD interaction (verteporfin). Values represent means ± SD. Statistical test: Mann-Whitney U. # - reference for statistical comparison, ***p<0.001. For (A), (B), and (C) actin was used as loading control.
Figure 5
Figure 5. YAP regulates Jag-1 in a β-catenin-independent and MST/Latsdependent manner
(A) Analyses of Jag-1, total YAP, nuclear YAP, phospho-YAP, TEAD4 (after IP with YAP antibody), Mst1, Mst2, and Hes-1 and corresponding relative proliferation of SNU-449 cells after combined inhibition of Mst1 and Mst2. SNU-449 cells were chosen because they show high-level expression of Mst1 and Mst2. Statistical analysis compares scramble siRNA and Mst1/2 siRNA. (B) Protein analyses of Jag-1, total YAP, nuclear YAP (after fractionation), phosphorylated YAP, TEAD4 (after IP with YAP antibody), Mst2, Lats2, and Hes-1 and corresponding cell proliferation of HuH-1 cells (showing low-levels of Mst2 and Lats2) after expression of Lats2 or Mst2. Statistical testing compares empty vector and Mst2 or Lats2, respectively. (C) Western blot analysis of YAP, Jag-1, Hes-1, and β-catenin in PLC/PRF/5 cells after overexpression of YAPwt and concomitant inhibition of β-catenin. (D) Analysis of β-catenin, OT, YAP, Jag-1, and Hes-1 in PLC/PRF/5 after overexpression of the constitutively active β-cateninS33Y mutant. For all Western blots, untreated (control), scramble siRNA-transfected, or empty vector-transfected cells were used as controls. Actin served as loading control. Values represent means ± SD. For statistical comparison, the Mann-Whitney U test was used. ***p<0.001.
Figure 6
Figure 6. Activation of YAP-induced Jag-1/Notch signaling correlates with poor prognosis of HCC patients
(A) Association between YAP and Jag-1 as well as between YAP and Hes-1 transcript levels in 27 primary human HCCs. The Spearman rank coefficient was used as a statistical measure of association. (B) Representative H/E staining and immunohistochemical analysis for YAP, Jag-1, Notch-1, CK19, and α-smooth muscle actin (SMA) in normal human liver and HCC (n=70). Original magnification: ×200. (C) Representative Western blot of cell lysates from human normal livers (NL; n=6), surrounding liver tissue (SL; n=48), HCC with better prognosis (HCC-B; n= 22), and HCC with poor prognosis (HCC-P; n=26). Protein analyses of Jag-1, NICD, total YAP, nuclear YAP (after fractionation), TEAD4, and Hes-1 were detected. (D) For analyzing the interaction between YAP and TEAD4 in primary HCC samples, YAP was immunoprecipitated (IP) and TEAD4 was detected by immunoblotting (IB). BP: negative control for IP. (E) Exemplary boxplot and statistical analyses of nuclear YAP and NICD in all NLs, SLs, HCC-B, and HCC-P after densitometric protein quantification. Actin and histone H3 were used for normalization of total protein (Jag-1, NICD, TEAD4, and Hes-1) and nuclear protein fractions (YAP), respectively. Open circles indicate outliers. Statistical test: Mann-Whitney U. ***p<0.001.
Figure 7
Figure 7. Concomitant activation of YAP/Jag-1 in pancreatic and colon cancer specimens
(A) Association between YAP and Jag-1 and between YAP and Hes-1 transcript levels in 11 human PDACs. (B) Representative H/E staining and immunohistochemical analysis for YAP and Jag-1 in pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) specimens. Original magnification: ×200. (C) Western blot analysis of YAP, Jag-1, and NICD in Panc-1 cells after siRNA-mediated silencing of YAP. Untreated (cont.) and scramble siRNA-transfected cells served as controls. Hes-1 transcript levels were determined by real-time PCR. (D) Viability kinetics of Panc-1 cells after inhibition of YAP measured by MTT-assay 48, 72, and 96 h after siRNA transfection for YAP (left) and Jag-1 (right). Untreated (cont.) and scramble-transfected cells served as controls. Values represent means ± SD. Statistical test: Mann-Whitney U; n.s. - not significant, **p<0.01. (E) Association between YAP and Jag-1 as well as between YAP and Hes-1 transcript levels in 40 human CRC samples. (F) Representative Western blots from human CRC tissue samples (n=40) and respective surrounding colon tissue (SC; n=40). Jag-1, NICD, total YAP, nuclear YAP (after fractionation), TEAD4, and Hes-1 were detected. Actin and histone H3 were used as loading controls for total protein and nuclear protein fractions, respectively. For (A) and (E) the Spearman rank coefficient was used as a statistical measure of association.
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