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p14/p19ARF (ARF) is a tumor suppressor gene that is frequently mutated in human cancer. ARF has multiple tumor suppressor functions, some of which are mediated by signalling to p53. Surprisingly, a significant fraction of human tumors retain persistently high levels of ARF, suggesting that ARF may possess a pro-survival function. We show that ARF protein is markedly upregulated in cells exposed to nutrient starvation. Cells with silenced ARF demonstrate reduced autophagy and reduced viability when placed under conditions of starvation. We show for the first time that ARF silencing can limit the progression of some tumors, such as lymphoma, but not others, such as E1A/ras-induced tumors. Specifically, myc-driven lymphomas with mutant p53 tend to overexpress ARF; we show that silencing ARF in these tumors greatly impedes their progression. These data are the first to show that ARF can act in a p53-independent manner to promote the progression of some tumors.
The ARF tumor suppressor is best known for its ability to stabilize p53, mediated by inhibition of the p53 ubiquitin ligase MDM2. Additionally, ARF has multiple tumor suppressor functions that are independent of p53: these involve interaction with a number of proteins with roles in cell proliferation, including nucleophosmin, NIAM, c-myc, E2F-1, DP1, and others (see 1 for review). Despite its multiple growth suppression functions, ARF is inexplicably overexpressed in a significant fraction of human tumors, including up to 50% of Burkitt’s lymphomas (2, 3), as well as the majority of tumors with mutant p53 (4). To date, the overwhelming majority of studies on ARF have focused on its tumor suppressor roles, and no groups have addressed the possibility that ARF might promote the survival of a subset of tumors.
A role for ARF in autophagy has recently been discovered. Specifically, a small molecular weight variant of this protein generated by translation from an internal methionine has been shown to localize to mitochondria and induce autophagy (5, 6). More recently, another group has shown that full-length ARF can likewise induce autophagy (7). Autophagy is a process of lysosome-mediated self-digestion that occurs during periods of nutrient deprivation (see 8 for review). This process is necessary for cell survival; following nutrient starvation proteins and organelles are degraded, and the released amino acids are utilized for the synthesis of essential proteins. Whereas recent studies have shown that ARF can induce autophagy, the relevance of ARF to starvation-induced autophagy, and the significance of this finding to cancer development, have not been addressed. In this study we show that nutrient deprivation leads to significant increases in ARF protein. We show that ARF-mediated autophagy can protect cells from periods of nutrient deprivation. Further, we show that lymphomas with ARF silenced demonstrate impaired progression in immunocompromised mice. Our data support the premise that ARF can enhance tumor development under certain circumstances, and that some tumors with mutant p53 may retain ARF in order to promote survival under metabolic stress.
Wild type, p53-null, and p53/ARF double knockout MEFs were cultured as described (9). B cell lymphomas B2998 and E330 were cultured in RPMI with 20% heat-inactivated fetal calf serum, 55 uM β-mercaptoethanol and 10 ng/mL IL-7 (R&D Systems). Retroviral infection, p53 short hairpins and short hairpin control vector are described (10). shRNA vectors for ARF were generated by PCR amplification of 97mer DNA oligonucleotides as described (11) and cloned into the LMP vector (12). Oligonucleotide sequences for shARF 157:
Unless otherwise indicated, shARF56 was used for all experiments to silence ARF. Cell viability assays were performed on using the ViaCount assay and the Guava PCA (Guava Technologies); statistical significance was calculated using the two-sided student’s t test.
Mitochondria were purified as described (13). Western blotting was performed on 100 ug whole cell lysate or 20 ug mitochondrial lysate; antisera used were anti-p19ARF (GeneTex), anti-actin (AC15, Sigma), anti-LC3 (Novus), and anti-Beclin-1 (Santa Cruz Biotechnology), anti-p62SQSTM1 (Santa Cruz Biotechnology). For electron microscopy, cells were fixed with 2% glutaraldehyde/2% formaldehyde in cacodylate buffer, fixed in 1% osmium tetroxide, embedded in epoxy resin and stained with uranyl acetate and lead citrate. The area of autophagosomes was calculated using NIH Image. Immuno-electron microscopy was performed as described (14) and visualized by secondary labeling with Protein A-colloidal gold. All samples were viewed in a FEI Tecnai 12 TEM operated at 80 kV.
Long-lived protein degradation was assayed as described (15). The percentage of long-lived protein degraded was obtained by the formula % degradation = (14C counts at timepoint/sum of 14C counts at each time point + total cell-associated radioactivity) × 100.
For xenograft analyses, MEFs were co-infected with retrovirus encoding luciferase along with retroviruses encoding vector, shBec1, shp53, or E1a/Ras before being injected (1×106 cells/injection) into SCID mice. E1A, Ras and luciferase levels were monitored by western analysis. Prior to injection, luciferase activity in infected cells was tested and silencing of all genes was confirmed by western analysis. For biophotonic imaging on the IVIS® Spectrum (Xenogen Biosciences), 1% isofluorane/oxygen-anesthetized mice were injected with luciferin (4 mg/animal) into the intraperitoneal cavity. In all cases the averaged data from each of two independent experiments using 3 animals for each sample were calculated; caliper measurements of tumors corroborated these analyses. For the lymphoma studies, cells were infected with shControl or shARF four times and selected in 1.5 ug/mL puromycin for 48 hours. After one week cells were resuspended in PBS and 1×106 cells were injected into the tail vein of SCID mice. For these studies the averaged data from each of two independent experiments (3 animals per sample) plus standard error of the mean were calculated and analyzed by the Wilcoxin two sample test, and p values were combined according to the method of Fisher.
ARF expression is negligible in normal non-transformed cells. This gene is also potently repressed by p53; consequently p53-null mouse embryo fibroblasts (MEFs) express high levels of ARF (4, 16). To determine the relevance of ARF to starvation-induced autophagy, we exposed p53-null MEFs to nutrient deprivation by incubating them in Hanks Buffered Salt Solution (HBSS), which is amino acid and growth factor-free. HBSS incubation led to a rapid (2 h) and significant up-regulation of ARF protein (Fig. 1A); this occurred independent of p53 status (Fig. 1B). Data quantitated from several experiments indicate that ARF is up-regulated approximately 1.6-fold in wt MEFs, and 6.5-fold in p53 −/− MEFs after 4 hours incubation in HBSS (data not shown). Induction of ARF mRNA by serum starvation has been noted previously (17). However, in response to HBSS, the up-regulation of ARF was not accompanied by an increase in ARF mRNA (data not shown). Further, there were no differences in ARF half-life under these conditions (Fig. 1C). The increase in ARF may be due to enhanced translation of ARF mRNA during nutrient deprivation.
ARF has been shown to play a role in autophagy via its localization to mitochondria (5). We could find no evidence for ARF localization at mitochondria in unstressed p53-null MEFs (Supplemental Fig. 1A); however, immuno-electron microscopy analyses revealed considerable localization of ARF to mitochondria after 2 hours of nutrient deprivation (Fig. 1D). These data were consistent with western analyses of purified mitochondria, which showed demonstrable ARF co-purifying with mitochondria after 2 hours of HBSS (Supplemental Fig. 1B). Quantitative western analysis and counting of grains from immuno-EM indicated that between five to ten percent of ARF localized to mitochondria following HBSS treatment.
The relevance of ARF upregulation to starvation-induced autophagy was assessed in p53-null MEFs using a short hairpin to silence this gene (shARF); this hairpin reduces over 90% of ARF protein, but does not silence p16ink4a expression (for example see Fig. 4A). In these experiments, the extent of autophagy was assessed by western analysis for the protein LC3, which becomes processed to a faster mobility species during autophagy (LC3 II), as well as by autophagosome formation and the analysis of long-lived protein degradation. Infection of p53-null MEFs with a parental retrovirus, or a virus encoding a short hairpin for p53 (which has no target in p53-null cells) did not influence the accumulation of LC3 II following HBSS treatment (Fig. 2A). Notably, however, ARF-silencing in p53-null MEFs led to markedly decreased accumulation of LC3 II following HBSS treatment (Fig. 2A). Moreover, electron microscopy indicated that following HBSS treatment, cells with silenced ARF had fewer, and often smaller, autophagosomes than control cells (Fig. 2B). The area of autophagosome coverage in HBSS-treated cells decreased from 48 +/− 5% to 7+/− 2% when ARF was silenced. Consistent with this, silencing ARF in p53-null MEFs reduced the degradation of long-lived proteins in response to HBSS treatment (Fig. 2C). Not surprisingly, this decrease in autophagy was accompanied by a decrease in viability; after 24 hours of nutrient deprivation in HBSS the majority of p53-null MEFs remained viable, while only 30% of cells with silenced ARF were viable at this timepoint (Fig. 2D). The combined data point to a previously unseen role for ARF in promoting cell survival during metabolic stress.
We next sought to determine whether ARF-mediated autophagy might promote tumor growth. For this analysis we chose to induce metabolic stress in tumors by silencing Beclin-1, a critical mediator of autophagy. Other groups have found that autophagy inhibition causes tumor regression (18, 19); similarly, we found that silencing Beclin-1 inhibited xenograft tumor development (Supplemental Fig. 3A). Interestingly, we found that the effect of Beclin-1 silencing on tumor growth was influenced by ARF status. For these studies we created luciferase-expressing transformed MEF cell lines that differed in ARF status (p53-null versus p53/ARF double knockout MEFs infected with E1A and Ras); these were subjected to Beclin-1 silencing and monitored for tumor development in a xenograft assay. We were able to achieve significant silencing of Beclin-1 in these lines (Fig. 3A). Bioluminescent imaging indicated that tumors with high ARF (p53-null) were consistently resistant to the growth suppressive effects of silencing Beclin-1, compared to tumors without ARF (p53/ARF double knockout, Fig. 3B, compare mouse 2A to 3A). Nearly identical results were obtained when we silenced ARF with a short hairpin (shARF, Fig. 3B, compare mouse 2A to 2B). The combined results from two independent experiments using three mice per virus are plotted in Figure 3C; these data indicate that tumors without ARF are markedly more sensitive to growth inhibition induced by Beclin-1 silencing.
To assess the physiological relevance of ARF-mediated autophagy to tumor development, we silenced ARF in tumors that overexpress this protein and assessed the impact on tumorigenesis. Toward this end we utilized a primary murine B cell lymphoma cell line (B2998) from the Eμ-myc transgenic mouse that contains mutant p53 (C.M. Eischen, unpublished results) and high levels of ARF. We infected this lymphoma cell line with one of two different short hairpins for ARF (shARF157 or shARF56) or a short hairpin control virus (shControl), followed by a luciferase retrovirus. Following confirmation that luciferase activity was equivalent and that ARF was silenced, cells were injected into the tail vein of immunocompromised mice and imaged after 7 days. As depicted in Figure 4A, infection with either of two different ARF short hairpins, but not with a short hairpin control, led to markedly impaired lymphoma development. This result was consistent in another lymphoma cell line containing mutant p53 (E330, Supplemental Fig. 3B), and was consistent when tumors were imaged after 7 or 14 days (data not shown). The combined data from two independent experiments using three mice per virus are plotted in Figure 4A; these data indicate that the inhibition of tumor progression by ARF silencing was reproducible and statistically significant (p< 0.01). Notably, ARF silencing also led to decreased viability of B2998 cells following HBSS treatment (Fig. 4B) and hypoxia (Supplemental Fig. 3C) and to decreased levels of steady state autophagy, as evidenced by accumulation of p62SQSTM1, which is degraded by autophagy (Fig. 4C). In contrast, ARF silencing did not affect the proliferation of lymphoma cells cultured under nutrient-rich conditions (Supplemental Fig. 3D). To confirm these findings in another cell line, we silenced ARF in a primary T cell lymphoma from the p53-null mouse; here again we found that ARF silencing reduced tumor development (Fig. 4D), suggesting that ARF can confer a survival advantage to certain tumors.
In this manuscript we provide data indicating that ARF has a previously undiscovered tumor-promoting activity, and that this activity may be mediated by its role in starvation-induced autophagy. These data do not discount the ample data indicating that ARF is a tumor suppressor, as all of our studies are performed in cells with mutant or null p53, exposed to metabolic stress. While some groups have reported that ARF-mediated autophagy is cytotoxic (5–7), we find that ARF is cytoprotective to tumors. This discrepancy may be due to differences in cell types analyzed, or the use of transfection and supra-physiologic overexpression of ARF by these other groups. We find that ARF-silencing in myc-driven lymphoma cells containing mutant p53 impedes their survival under nutrient-starved conditions and their development as xenografts. These data are the first to indicate that the ARF tumor suppressor has a survival function, and further that this survival function may be selected for in certain tumors. It should be noted, however, that this survival function is likely to be tumor-type specific; consistent with the data of others (9), we find that ARF silencing enhances the development of E1A/Ras-driven tumors (Fig. 3C). Ras can inhibit autophagy (20), and this may explain why ARF-mediated autophagy has no selective benefit to Ras-driven tumors. The findings described here-in have important clinical relevance: autophagy modulators like chloroquine and rapamycin are currently used in clinical trials for human cancer. Our data suggest that tumors without ARF may be more susceptible to such treatment.
The authors thank Gerry Zambetti, Scott Lowe and Shengkan Jin for reagents. We thank Eileen White and Carol Prives for critical reading of the manuscript, and Cory Abate-Shen for communicating results prior to publication. This work was supported by NIH R01 CA080854 and CA150002 (M.M.), and by the Philippe Foundation (O.H).