Volume 137, Issue 5, Pages 1805-1815.e4 (November 2009) Amphiregulin Induces the Alternative Splicing of p73 Into Its Oncogenic Isoform ΔEx2p73 in Human Hepatocellular Tumors  Josefa Castillo, Saioa Goñi, María Ujue Latasa, María J. Perugorría, Alicia Calvo, Jordi Muntané, Paulette Bioulac–Sage, Charles Balabaud, Jesús Prieto, Matías A. Avila, Carmen Berasain  Gastroenterology  Volume 137, Issue 5, Pages 1805-1815.e4 (November 2009) DOI: 10.1053/j.gastro.2009.07.065 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 ΔEx2p73 is expressed in chronic liver disease and HCC. (A) Prevalence of ΔEx2p73 expression in healthy liver tissue (HL, n = 21), healthy liver tissue from organs harboring a hepatocellular carcinoma (HCC) (HL+HCC, n = 13), cirrhotic liver tissue (Cirr, n = 15), cirrhotic liver tissue from organs harboring an HCC (Cirr+HCC, n = 11), and HCC tissues (n = 15). **P < .01 and ***P < .001 vs HL. (B, C, and D) Quantification of TAp73, ΔEx2p73, and PEDF mRNA levels in the different groups of samples described above. *P < .05, **P < .01, and ***P < .001 vs HL. (E and F) Multiplex PCR analyses of TAp73 and ΔEx2p73 mRNAs in representative samples from the indicated types of tissues and human HCC cell lines. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 AR treatment regulates ΔEx2p73 mRNA levels in human HCC cells. (A) Quantification of TAp73 and ΔEx2p73 mRNAs in AR-treated Hep3B cells (4 hours). Values are relative to untreated cells. (B) Dose-dependent effect of AR (4 hours) on ΔEx2p73/TAp73 mRNA ratio in Hep3B cells. *P < .05 vs untreated cells (C). (C) Effect of AR on ΔEx2p73/TAp73 mRNA ratio in isolated human hepatocytes. *P < .05 vs untreated cells (C). (D) Inhibition of AR (4 hours) effect on ΔEx2p73/TAp73 mRNA ratio in Hep3B cells by the EGFR inhibitor PD153035 (30-minute preincubation) (AR+PD) and Western blot of phospho-EGFR in AR-treated cells (12 minutes) with or without PD153035 (30-minute preincubation) (AR+PD). (E) Effect of AR (4 hours) on PUMA and PEDF mRNA levels with or without PD153035 (30-minute preincubation) (AR+PD) in Hep3B cells. Values are relative to untreated control cells. *P < .05 vs untreated cells (C), #P < .05 vs AR treated cells. (F) Disruption of AR autocrine or paracrine loop lowers the ΔEx2p73/TAp73 mRNA ratio. Treatment of PLC/PRF/5 human HCC cells with AR-neutralizing antibodies (20 μg/mL), the ADAM17 inhibitor TAPI-1, or the EGFR inhibitor PD153035 for 7 hours after 48 hours starvation inhibits the accumulation of ΔEx2p73 transcripts. Values are relative to those found in control cells incubated with vehicle (DMSO) for TAPI-1 and PD153035 or purified goat IgG (20 μg/mL) for AR-neutralizing antibodies. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 AR does not affect ΔEx2p73 mRNA stability. (A) The effect of AR on ΔEx2p73/TAp73 mRNA ratio is lost in Hep3B cells preincubated with ActD (1 μg/mL, 3 hours) and treated with AR (4 hours). *P < .05 vs untreated cells (C). (B) ΔEx2p73 mRNA half-life was not affected by AR treatment. Hep3B cells were incubated with ActD as above in the presence or absence of AR for up to 7 hours. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 AR produced by HCC cells promotes p73 alternative splicing and ΔEx2p73 expression. (A) Knockdown of AR expression results in significant loss of cell viability, as observed 72 hours after transfection of PLC/PRF/5 cells with control (siGL) or AR specific (siAR) siRNAs. (B) Expression of TAp73 and ΔEx2p73 mRNAs in PLC/PRF/5 cells 72 hours after AR-specific siRNA transfection. (C) Knockdown of endogenous AR expression reduces ΔEx2p73 mRNA levels in human HCC cell lines. AR and ΔEx2p73 mRNAs were quantitated 72 hours after transfections. * P < .05 and **P < .01 vs controls. (D) Inhibition of endogenous AR expression results in the up-regulation of the p73 target genes PEDF and PUMA. Gene expression was analyzed 72 hours after transfections. *P < .05 vs controls. All values are referred to expression levels found in transfections with control siRNA (siGL). Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Signaling of AR effect on p73 alternative splicing and ΔEx2p73 expression is mediated by JNK1. (A) Effect of down-regulation of endogenous AR expression (left panels) or AR treatment (right panels) on JNK1, JNK2, and PI3K/Akt phosphorylation in PLC/PRF/5 cells. Western blots were performed on samples obtained 72 hours after transfections or 12 minutes after AR treatment. (B) Specific knockdown of JNK1, JNK2, and PI3K in PLC/PRF/5 cells was validated at the mRNA level (upper panel) and by Western blotting (lower panel). Samples were analyzed 72 hours after transfections. *P < .05 vs siGL. (C) PLC/PRF/5 cells were transfected with siRNAs specific for JNK1, JNK2, PI3K, or control siRNA (siGL) and, after 72 hours, were treated with AR (4 hours). ΔEx2p73/TAp73 mRNA ratios are shown. *P < .05 vs cells transfected with siGL and treated with AR. (D) ΔEx2p73/TAp73 mRNA ratio in PLC/PRF/5 cells 72 hours after transfection with JNK1 specific siRNA. *P < .05 vs siGL. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 JNK1 activation by AR inhibits Slu7 expression. (A) AR treatment (12 minutes) activates Elk-1 phosphorylation in PLC/PRF/5 cells. This effect was blocked by the EGFR inhibitor PD153035 (30-minute preincubation). Basal Elk-1 phosphorylation in PLC/PRF/5 cells 72 hours after transfection with AR specific (siAR) or control (siGL) siRNAs are shown. (B) Box plots showing Slu7 mRNA levels in the different groups of liver tissue samples described in Figure 1. ***P < .001 vs HL. (C) Effect of AR (4 hours) treatment on Slu7 mRNA levels in cultured human hepatocytes and in PLC/PRF/5 cells 72 hours after transfection with control or JNK1 specific siRNAs. *P < .05 vs controls (C), #P < .05 vs siGL plus AR. (D) AR-stimulated association of Elk-1 with the Slu7 promoter. ChiP was performed in Hep3B cells treated with AR (50 nmol/L, 4 hours). Graph shows the quantification by real-time PCR of immunoprecipitated Slu7 promoter DNA fragments. Inset shows ethidium bromide-stained gels. (E) PLC/PRF/5 cells were transfected with AR specific or control (siGL) siRNAs, and Slu7 mRNA levels were determined 72 hours after transfections. **P < .01 vs siGL alone. (F) PLC/PRF/5 cells were transfected with Slu7-specific (siSlu7) or control siGL (C) siRNAs, and, 96 hours later, levels of Slu7, ΔEx2p73, and TAp73 mRNAs were determined. *P < .05 vs respective controls. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Model for the regulation of p73 pre-mRNA alternative splicing by AR. (A) In the absence of AR, normal cellular levels of Slu7 would favor exon inclusion and the correct splicing of p73 pre-mRNA into TAp73 mRNA. TAp73 protein may then regulate the expression of its target genes such as PUMA and PEDF. (B) Upon AR up-regulation, binding and activation of the EGFR triggers an intracellular signaling cascade leading to JNK1 activation and Elk-1 phosphorylation. Activated Elk-1 can repress Slu7 gene promoter, resulting in insufficient Slu7 protein levels to mediate the correct splicing of p73 pre-mRNA. The alternative spliced ΔEx2p73 variant behaves as a dominant negative agent towards TAp73 and p53. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure S1 (A and B) Quantification of PAP-2a and AQP3 expression in healthy liver tissue (HL, n = 21), healthy liver tissue from organs harboring an HCC (HL+HCC, n = 13), cirrhotic liver tissue (Cirr, n = 24), cirrhotic liver tissue from organs harboring an HCC (Cirr+HCC, n = 11), and HCC tissues (n = 15). ***P < .001 vs HL. The expression of PAP2a and AQP3 showed a statistically significant negative correlation with that of ΔEx2p73 when analyzed in the above-mentioned groups of samples (r = −0.68, P < .001 and r = −0.32, P < .05 for AQP3 and PAP2a, respectively). (C) Effect of AR (4 hours) treatment on PAP2a and AQP3 messenger RNA (mRNA) levels in Hep3B cells. Values are relative to untreated controls (C). *P < .05 vs controls. (D) Expression of PAP2a and AQP3 in PLC/PRF/5 cells after 72-hour transfection with AR specific siRNA (siAR). Values are relative to cells transfected with control siRNA (siGL). *P < .05 vs controls. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure S2 (A and B) Dose-dependent effect of AR (4 hours) treatment on ΔEx2p73/TAp73 mRNA ratio in SK-Hep1 (A) and PLC/PRF/5 (B) cells. *P < .05 vs untreated cells (C). (C) Effect of AR overexpression on the ΔEx2p73/TAp73 ratio. SK-Hep1 cells were transfected with an AR-expressing vector (pcV5-AR) or with the same empty vector (pcV5). ΔEx2p73 and TAp73 mRNA levels were measured 24 hours after transfections. *P < .05 vs cells transfected with control plasmid. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure S3 Effect of JNK1 and JNK2 overexpression on AR-stimulated TAp73 splicing. Hep3B cells were transfected with either JNK1 or JNK2 expression vectors or with a control empty vector (pcDNA). Twenty-four hours after transfection, cells were treated with AR for 4 hours, and ΔEx2p73 and TAp73 mRNA levels were measured. In cells overexpressing JNK1, the effect of AR on the ΔEx2p73/TAp73 ratio was significantly enhanced (*P < .05 vs control, pcDNA transfected cells). JNK2 transfection had no effect. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure S4 (A and B) Effect of AR overexpression on Slu7; ΔEx2p73/TAp73 ratio; and the expression of PEDF, PUMA, PAP2a, and AQP3 in HepaRG (A) and THLE3 (B) nontumorigenic hepatocyte human cell lines. Cells were transfected with an AR expression vector (pcV5-AR) or a control vector (pcV5), and the expression of the above-mentioned genes was measured 48 hours after transfections. *P < .05 vs pcV5 transfected cells. (C and D) Effect of ΔEx2p73 expression on TAp73 target genes in HepaRG and THLE3 cells. Cells were transfected with a ΔEx2p73 expression vector (pcV5-ΔEx2) or a control vector (pcV5), and the expression of TAp73 target genes was measured 48 hours after transfections. *P < .05 vs pcV5 transfected cells. Gastroenterology 2009 137, 1805-1815.e4DOI: (10.1053/j.gastro.2009.07.065) Copyright © 2009 AGA Institute Terms and Conditions