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. 2008 Oct;135(4):1322-32.
doi: 10.1053/j.gastro.2008.07.012. Epub 2008 Jul 17.

E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR and COX-2 in a mouse model of human liver cancer

Sara Ladu  1 , Diego F Calvisi, Elizabeth A Conner, Miriam Farina, Valentina M Factor, Snorri S Thorgeirsson
Affiliations

E2F1 inhibits c-Myc-driven apoptosis via PIK3CA/Akt/mTOR and COX-2 in a mouse model of human liver cancer

Sara Ladu et al. Gastroenterology. 2008 Oct.

Abstract

Background & aims: Resistance to apoptosis is essential for cancer growth. We previously reported that hepatic coexpression of c-Myc and E2F1, 2 key regulators of proliferation and apoptosis, enhanced hepatocellular carcinoma (HCC) development in transgenic mice. Here, we investigated the molecular mechanisms underlying oncogenic cooperation between c-Myc and E2F1 in relationship to human liver cancer.

Methods: Activation of pro- and antiapoptotic cascades was assessed by immunoblotting in experimental HCC models and in human HCC. Effect of antisense oligodeoxy nucleotides against c-Myc and E2F1 was studied in human HCC cell lines. Suppression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PIK3CA)/Akt, mammalian _target of rapamycin (mTOR), and cyclooxygenase (COX)-2 pathways was achieved by pharmacologic inhibitors and small interfering RNA in human and mouse HCC cell lines.

Results: Coexpression with E2F1 did not increase proliferation triggered by c-Myc overexpression but conferred a strong resistance to c-Myc-initiated apoptosis via concomitant induction of PIK3CA/Akt/mTOR and c-Myb/COX-2 survival pathways. COX-2 was not induced in c-Myc and rarely in E2F1 tumors. In human HCC, PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways were similarly activated, with levels of PIK3CA/Akt, mTOR, and c-Myb being inversely associated with patients' survival length. Silencing c-Myc and E2F1 reduced PIK3CA/Akt and mTOR and completely abolished c-Myb and COX-2 expression in human HCC cell lines. Finally, simultaneous inhibition of PIK3CA/Akt/mTOR and COX-2 activity in in vitro models caused massive apoptosis of neoplastic hepatocytes.

Conclusions: E2F1 may function as a critical antiapoptotic factor both in human and in rodent liver cancer through its ability to counteract c-Myc-driven apoptosis via activation of PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways.

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Figures

Figure 1
Figure 1
Labeling (A) and apoptotic (B) indices in dysplastic livers and HCC (C) developed in E2F1, c-Myc, and c-Myc/E2F1 mice. The same specimens were analyzed for labeling index (percentage of PCNA-positive cells in 2000 cells) and apoptotic index (percentage of terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL)-positive cells in 2000 cells). Means and 95% confidence intervals from 10 tumors for each genotype are shown. In (A), a, P=0.002; b, P<2.81E-05; c, P<2.41E-05 versus c-Myc and c-Myc/E2F1; in (B), a, P=0.0001 versus E2F1; b, P=0.004 versus c-Myc/E2F1; c, P=0.007 versus E2F1; d, P=9.50E-07 versus E2F1; e, P=0.01 versus c-Myc/E2F1; f, P=0.004 versus E2F1; g, P=8.97E-05 versus E2F1; h, P=0.001 versus c-Myc/E2F1; i, P=0.009 versus E2F1; in (C); a, P=1.38E-20 versus c-Myc; b, P=7.02E-11 versus E2F1; c, P=1.61E-08 versus c-Myc; d, P<5.44E-06 versus c-Myc; and e, P<3.34E-06 versus c-Myc/E2F1 by two-sided Student’s t test.
Figure 2
Figure 2
Activation of Akt (A) and mTOR (B) cascades in c-Myc, E2F1, and c-Myc/E2F1 hepatic lesions. Whole cell lysates were prepared from wild-type livers (Wt), dysplastic livers (D) and HCC (T) from at least 15 samples for each transgenic line and immunoblotted with indicated antibodies. Representative western blot is shown. (C) Densitometric analysis of c-Myc and E2F1 levels. Optical densities were normalized to β-actin values and expressed in arbitrary units. Each bar represents mean±SD. Horizontal lines show the levels of c-Myc and E2F1 proteins in wild type mice.
Figure 3
Figure 3
Activation of COX-2 and downstream effectors in wild-type livers (Wt), dysplastic livers (D) and HCC (T) from c-Myc, E2F1, and c-Myc/E2F1 transgenic mice .(A). Whole cell lysates were prepared from at least 15 samples per each stage (dysplasia, HCC) for each transgenic line and immunoblotted with indicated antibodies. Representative western blot is shown. (B) Densitometric analysis of COX-2 and downstream effectors. Optical densities were normalized to β-actin values and expressed in arbitrary units. Each bar represents mean±SD.
Figure 4
Figure 4
Activation of Akt and mTOR (A) and COX-2 (B) in human liver samples. Whole cell lysates were prepared from normal livers (NL), surrounding livers (SL) and HCC with better (b) or poor (p) prognosis and immunoblotted with indicated antibodies. Equal protein loading was verified by β-actin expression. 25 tissue samples were analyzed from each HCC group and matching surrounding livers, and 5 tissue samples from normal livers. Representative western blot analysis is shown.
Figure 5
Figure 5
Effect of antisense oligodeoxy nucleotides against c-Myc (A), E2F1 (B), c-Myc and E2F1 (C), and c-Myb (D) on the expression of Akt, mTOR, c-Myb, and COX-2 in Alexander HCC cell line. Cells were subjected to serum deprivation for 24 h, followed by treatment with antisense oligodeoxy nucleotides (AODN) against c-Myc, E2F1, and c-Myb. Experiments were repeated at least 3 times, the results were reproduced in HuH1 and HuH7 HCC cell lines. AODN, antisense oligodeoxy nucleotides; SCR, scramble.
Figure 6
Figure 6
Effect of inhibition of Akt, mTOR and COX-2 pathways in vitro and in vivo. Human (Alexander, HuH1, and HuH7) and mouse (E2F1_1 and E2F1_2) HCC cell lines were treated with Akt inhibitor LY294002 (50 µM), mTOR inhibitor Rapamycin (10 nM), PIK3CA/mTOR inhibitor PI-103 (0.5 µM), and COX-2 inhibitor CAY10404 (50 µM) for 24 h. (A and C) Cell viability was determined by WST-1 assay performed in a 96-well format as described in the Materials and Methods (B and D). Apoptosis was determined by Cell Death Detection Elisa Plus kit performed in a 96-well format as described in the Materials and Methods. Values for apoptosis and cell viability represent the mean and 95% confidence interval from 3 independent duplicate experiments.
Figure 7
Figure 7
Overview of signal transduction pathways triggered by hepatic co-expression of c-Myc and E2F1 protooncogenes. Overexpression of c-Myc alone results in induction of apoptosis via the ASK1/JNK/p38MAPK cascade and activation of translation via elF4E. Overexpression of E2F1 upregulates Akt and mTOR pathways leading both to inhibition of apoptosis through suppression of the ASK1/JNK/p38MAPK axis and activation of translation via inhibition of 4EBP1 and activation of elF4G. mTOR activation might depend on Akt activation and/or other undefined mechanisms (question mark). Co-expression of c-Myc and E2F1 results in suppression of c-Myc-driven apoptosis and transactivation of the c-Myb protooncogene leading to a strong induction of the COX-2 survival signaling. Once upregulated, COX-2 promotes cell survival via activation of the anti-apoptotic proteins Bcl-2 and MCl-1, and by inhibiting the TRAIL-dependent apoptosis via downregulation of DR5. Resistance to TRAIL-dependent apoptosis is further achieved via promoter methylation of the TRAIL effector DAPK.
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