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Cancer Research 68, 6049, August 1, 2008. doi: 10.1158/0008-5472.CAN-08-1279
© 2008 American Association for Cancer Research
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COOH-Terminal Binding Protein Regulates Expression of the p16INK4A Tumor Suppressor and Senescence in Primary Human Cells

Edmund A. Mroz1, Abigail H. Baird1, William A. Michaud1 and James W. Rocco1,2

1 Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital Cancer Center and Harvard Medical School and 2 Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts

Requests for reprints: James W. Rocco, Massachusetts General Hospital Cancer Center, Jackson 904, 55 Fruit Street, Boston, MA 02114. Phone: 617-726-5251; Fax: 617-726-8623; E-mail: jrocco{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The p16/pocket-protein pathway sets a balance between tumor suppression and capacity for tissue regeneration. Understanding the upstream signaling pathway that turns on the expression of p16 is required both for knowing the tumorigenic stresses from which this pathway provides protection and for appreciating the selective pressure that leads to the loss of this pathway in most human tumors. We report that COOH-terminal binding protein (CtBP), a physiologically regulated transcriptional corepressor that dimerizes to hold together repressive complexes, regulates p16 expression in primary human fibroblasts and keratinocytes. Interfering with CtBP-mediated repression increased p16 expression and accelerated senescence. CtBP had little influence on the expression of the alternate product of the CDKN2A tumor-suppressor gene, p14ARF. Loss of CtBP-mediated repression diminished the Polycomb-based epigenetic histone mark that is reported to favor silencing of p16 via DNA methylation. Enhancing CtBP-mediated repression by growing cells in low oxygen increased the association of CtBP with the p16 promoter, as assessed by chromatin immunoprecipitation, and reduced p16 expression. Stresses and stimuli that reduce CtBP-mediated repression are associated with increased p16 expression; therefore, CtBP may provide a common final target for regulating the balance among tumor suppression, regenerative capacity, and senescence. [Cancer Res 2008;68(15):6049–53]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The p16INK4A tumor suppressor protein (p16) inhibits the hyperphosphorylation of retinoblastoma-related pocket proteins that is required for cell cycle progression. It thus prevents inappropriate division of stressed cells and can lead to permanent exit from the cell cycle via senescence (1, 2). This critical tumor-suppressor role of p16 comes at a significant cost to the organism: decreased replicative potential of stem cells required for normal regeneration of many tissues (1).

Although effector mechanisms downstream from p16 are reasonably well understood, upstream mechanisms that couple stresses and stimuli to increased p16 expression are less well characterized (1, 2). Recent work has emphasized Polycomb-based repression of p16, centered on epigenetic trimethylation of lysine 27 in histone H3 (H3K27me3) at the CDKN2A gene that codes for p16 (3, 4). Mechanisms that couple signals to loss of epigenetic repression and to increased p16 expression, however, have not been identified. Few mechanisms that regulate p16 separately from the alternate product of the CDKN2A gene, p14ARF, are known.

Our comparison of the oncoproteins adenoviral E1A and the large T antigen of SV40 pointed us to a previously unsuspected component of upstream p16 control—COOH-terminal binding protein (CtBP), a physiologically regulated corepressor (5, 6). CtBP dimers form bridges between proteins having PxDLS amino acid motifs, including several transcription factors and other proteins involved in the regulation of transcription, including CtIP and components of Polycomb complexes. Several pathways regulate CtBP-mediated repression via effects on dimerization, nuclear localization, or degradation. Notably, oxidative stress, UV exposure, and wound healing, which are associated with increased p16 expression (1, 7), involve pathways that relieve CtBP-mediated repression (5, 6). Our results document the role of CtBP in the control of p16 and indicate how CtBP-mediated repression could contribute to the silencing of p16 in the hypoxic environment typical of solid tumors (8).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. Fibroblasts and keratinocytes were prepared from discarded human foreskins. Fibroblasts, cultured in DMEM containing 10% fetal bovine serum, penicillin, and streptomycin, were typically used 20 population doublings after the initial preparation (PD 20). Keratinocytes were cultured in fully supplemented keratinocyte serum-free medium (7). "Hypoxia" was the culture of cells in controlled 1% oxygen (BNP-210 incubator; ESPEC North America) instead of in normal atmospheric (21%) oxygen.

Expression and short hairpin constructs. Expression constructs for E1A mutants were derived by PCR from pLPC-12S E1A, a gift from S. Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); those based on the two human CtBP proteins, CtBP1 and CtBP2, were derived from gifts from G. Chinnadurai (Institute for Molecular Virology, Saint Louis University, St. Louis, MO). Short hairpin sequences (targeting both CtBP1 and CtBP2; sequences available on request) were constructed in the pBS-U6 vector (gift of Y. Shi, Department of Pathology, Harvard Medical School, Boston, MA) and subcloned into vectors based on the pLL 3.7 plasmid. All constructs were verified by sequencing. To allow selection for lentivirally infected cells, the enhanced green fluorescent protein coding sequence in pLL 3.7 was replaced with sequences coding for puromycin or neomycin resistance.

Virus production and infection. Replication-defective retroviruses and lentiviruses were produced by cotransfection of pLPC- or pLL-based plasmids with helper plasmids into 293T cells; control virus from empty vectors was generated in parallel. Infections were via spin infection at 1,500 x g for 1 h at room temperature in the presence of 8 µg/mL polybrene. Retroviral or lentiviral infections were used to introduce expression constructs or short hairpin RNA (shRNA) constructs, respectively, following the protocol in Fig. 1A . Puromycin at 1 to 5 µg/mL or G418 at 500 to 1,000 µg/mL was used for selection.


Figure 1
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  		Figure 1. The E1A oncoprotein induced p16 expression in primary human fibroblasts by interacting with the corepressor CtBP. A, although both oncoproteins have LxCxE motifs that interfere with pocket-protein function, expression of 12S E1A, unlike the large T antigen of SV40, increased p16 protein (Western blot, top left) and mRNA (quantitative PCR results, top right; relative to expression in vector control cells). Bottom, diagram of standard experimental protocol. B, diagram of conserved regions of E1A (10) and of expression constructs used in this study. Constructs were based on 12S E1A, which lacks CR3 of the 13S E1A protein. The 12S{Delta}CR4 construct lacked 8 amino acids centered on the CtBP-interacting PLDLS motif, but included the first 21 amino acids of CR4 and the nuclear localization signal at the COOH terminus. C, the increased p16 expression involved the part of E1A that interacts with CtBP (Western blot). 12S{Delta}CR4, lacking CtBP interaction, was deficient at stimulating p16 expression.

 
Protein analysis. NP40 lysis buffer extracts were separated on NuPage (Invitrogen) SDS-polyacrylamide gradient gels and transferred to polyvinylidene difluoride membranes. Antibodies for Western blotting were as follows: anti-CtBP, sc-17759 (recognizes both CtBP1 and CtBP2; Santa Cruz Biotechnology); anti-E1A, monoclonal antibody M58 (recognizes a region shared among all the E1A mutants used in this study; Massachusetts General Hospital Cancer Center); anti-p16, monoclonal antibody JC8 (a gift from James Koh, Department of Surgery, Duke University Medical Center, Durham, NC); and SV40 large T antigen, monoclonal antibody PAB419 (Massachusetts General Hospital Cancer Center). Western blots were visualized with chemiluminescence.

mRNA analysis. RNA was prepared from TRIzol (Invitrogen) extracts, followed by reverse transcription into cDNA with random primers and Superscript II (Invitrogen). Primers for PCR amplification of cDNA were designed so that products spanned exon breaks. Primer sequences are available on request.

Chromatin immunoprecipitation. The EZ-ChIP protocol (Upstate) was used. Antibodies were as follows: anti-CtBP, sc-11390 (Santa Cruz Biotechnology); anti-histone H3K4me3, ab8580 (Abcam); and anti-histone H3K27me3, 07-449 (Upstate). Chromatin bound to these polyclonal rabbit antisera was precipitated with protein A-agarose.

Senescence-associated β-galactosidase. Glutaraldehyde-fixed cells were incubated with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside substrate (pH 6) at 37°C (9).

Promoter-reporter studies. A 2.3-kb fragment of human genomic DNA preceding the start of the p16 coding sequence was cloned into the pGL3-basic vector (Promega) upstream of the vector's firefly luciferase coding sequence to produce pGL3-p16. pLPC-based expression plasmids were cotransfected with pGL3 reporter plasmids and with CMV-renilla luciferase (transfection control) into U2OS cells. Total transfected DNA was kept constant by adding empty pLPC.

Real-time PCR. QuantiTect SYBR Green Master Mix (Qiagen) or HotStart-IT Taq Master Mix (U.S. Biochemical; supplemented with SYBR Green, Invitrogen) was used in an Opticon real-time PCR instrument (Bio-Rad). PCR products were verified by melting curves and by gel electrophoresis. Genomic DNA provided standards for quantitative analysis of chromatin immunoprecipitation (ChIP) samples. Results for immunoprecipitated DNA are expressed as percentages of the DNA in an input sample representing 1/10th of the chromatin subjected to immunoprecipitation. For cDNA analysis, samples being compared with any one primer set were analyzed together. Relative amounts of cDNA between two samples within a primer set are expressed proportionate to 2{Delta}Ct. All quantitative PCR results are presented as the mean ± SE of triplicate determinations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
The E1A oncoprotein increased p16 expression through its interaction with CtBP. We found that the adenoviral E1A and the SV40 large T oncoproteins differed substantially in the ability to increase p16 in human fibroblasts. Five days after transduction, fibroblasts expressing the 12S form of E1A had substantially increased p16 protein and mRNA levels, whereas those expressing large T antigen showed little change in p16 (Fig. 1A). Because both oncoproteins contain LxCxE motifs that interfere with pocket-protein function (10, 11), this result suggested that a function of E1A separate from its effects on pocket proteins was responsible for increasing p16 at this early time after expression.

To identify the functions of E1A responsible for increasing p16 expression, we took advantage of the well-defined interactions of E1A with host-cell proteins (10) and compared a panel of 12S E1A mutants (Fig. 1B) for the ability to turn on p16 expression (Fig. 1C). The mutant lacking conserved region 1 (CR1) of E1A was essentially as effective as 12S E1A. Because 12S E1A lacks CR3 of full-length E1A, we concluded that neither CR1 nor CR3 provides functions necessary for p16 induction. Cells expressing 12S {Delta}CR2, without the LxCxE motif, also had elevated p16, supporting the hypothesis that interference with pocket proteins was not required to increase p16 expression. Cells expressing 12S {Delta}CR2, however, ceased dividing and took on morphologic characteristics of senescence, consistent with the intact downstream effects of p16 (Supplementary Fig. S1).

In contrast, the mutant called 12S {Delta}CR4, lacking only eight amino acids centered on the CtBP-interacting PLDLS motif, was very deficient at increasing p16 although nuclear localization of this mutant was intact (Fig. 1C; Supplementary Fig. S2). This result suggested that binding of E1A to CtBP was critical for the induction of p16 and that CtBP might normally play a role in regulating p16 expression through repression.

Knockdown of CtBP increased p16 in human fibroblasts and keratinocytes and accelerated senescence. As an independent test of whether CtBP mediates the repression of p16, we knocked down CtBP. Either of the two shRNA constructs targeting both human CtBP proteins led to elevated p16 in primary human fibroblasts (Fig. 2A ), an effect countered by CtBP expression constructs resistant to the shRNA (Supplementary Fig. S3). CtBP knockdown also increased p16 in primary human keratinocytes (Fig. 2B), showing that CtBP can regulate p16 expression in both epithelial and mesenchymal primary human cells.


Figure 2
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  		Figure 2. CtBP mediates the repression of p16 expression and senescence in primary human cells. A, CtBP knockdown increased p16 in primary human fibroblasts. Western blots of extracts taken 3 d after infection with control lentivirus or lentivirus coding for shRNAs (shRNA-a and shRNA-b) targeting CtBP. No drug selection. B, CtBP knockdown increased p16 in primary human keratinocytes. Western blots of extracts taken 1 wk after infection. C, prior CtBP knockdown ({blacksquare}) slowed the proliferation of PD 20 human fibroblasts (control, {blacktriangleup}). At day 0, equal numbers of cells were plated 5 d after CtBP knockdown or control lentiviral infection. Points, mean cell number (n = 4); bars, SE. D, CtBP knockdown (CtBP-kd) accelerated senescence of PD 40 human fibroblasts. Light micrographs of fibroblasts stained for senescence-associated β-galactosidase activity (blue) 10 d after infection.

 
Interference with CtBP-mediated repression had effects consistent with the elevated p16 protein levels, slowing cellular proliferation and accelerating the senescence that normally develops with the passage of primary cells in culture (2). Knockdown of CtBP with shRNA slowed a proliferation of fibroblasts even in mid-passage cells far from senescence (Fig. 2C). In later-passage fibroblasts, still 15 population doublings before their usual senescence with passage in culture, CtBP knockdown induced growth arrest along with the morphologic and histochemical changes of cellular senescence (Fig. 2D).

CtBP repressed the expression of p16 from the CDKN2A gene, with little effect on the alternate gene product p14ARF. Using several methods, we determined that CtBP affected transcription, specifically of p16 mRNA, from the CDKN2A gene. First, CtBP knockdown increased p16 mRNA while having little effect on mRNA for the alternate product of the CDKN2A gene, p14ARF (Fig. 3A ). Second, exogenous CtBP repressed the transcription of a luciferase reporter gene controlled by a p16 promoter. Coexpression of 12S-E1A countered this repression, as expected for CtBP-mediated repression (Fig. 3B). Third, CtBP knockdown affected epigenetic marks at the p16 promoter in a way consistent with transcriptional activation. As reported for embryonic stem cells and murine embryonic fibroblasts (12, 13), the p16 promoter in primary human fibroblasts had "bivalent" epigenetic covalent modifications of its associated histone H3, with both the activation-related trimethylation of lysine 4 (H3K4me3) and the repression-related trimethylation of lysine 27 (H3K27me3). In primary human fibroblasts, CtBP knockdown reduced the repression-related H3K27me3 epigenetic mark at the p16 promoter while having little effect on the activation-related H3K4me3 mark (Fig. 3C). This resolution of the epigenetic marking of the p16 promoter into the activated form is expected if CtBP knockdown increases the transcription of p16 mRNA from the CDKN2A gene.


Figure 3
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  		Figure 3. CtBP affected transcription from the p16 promoter of the CDKN2A gene. A, CtBP knockdown (CtBP-kd) for 5 d increased p16 mRNA but had little effect on p14ARF mRNA. No drug selection. Quantitative PCR of cDNA. Columns, mean of experimental/control ratio; bars, SE (CtBP-kd/vector). B, CtBP repressed and E1A derepressed expression from a firefly luciferase p16 promoter reporter. pGL3-basic (no upstream promoter) or pGL3-p16 (upstream human p16 promoter) was transfected into U2OS cells along with the indicated expression constructs and CMV-Renilla luciferase as a transfection control. Results are ratios of firefly to Renilla luciferase activity, with the activity ratio in cells transfected with pGL3-basic and empty vector taken as 1. C, CtBP knockdown resolved the p16 promoter in human fibroblasts from a bivalent to an activated epigenetic state. Quantitative ChIP with antibodies against histone H3 trimethylated at lysine 27 (H3K27me3) or at lysine 4 (H3K4me3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was used as control for an expressed gene.

 
Hypoxia enhanced the association of CtBP with the p16 promoter and diminished p16 expression. Consistent with its role as a transcriptional corepressor, we found that CtBP associated physically with genomic DNA near the p16 transcription start site (Fig. 4A ). To test the physiologic significance of this association, we performed cell cultures in hypoxia. Hypoxia enhances CtBP-mediated repression via an increase in the nuclear NADH/NAD+ ratio, which favors the formation of repressive CtBP dimers (5, 6). Hypoxia enhanced the association of CtBP with the p16 promoter; p16 mRNA and protein levels decreased correspondingly (Fig. 4B and C). CtBP knockdown removed this effect of hypoxia on p16 expression, leading to similar p16 levels regardless of oxygenation (Fig. 4C), indicating that CtBP directly mediates this effect of ambient oxygen on p16 expression.


Figure 4
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  		Figure 4. Hypoxia enhanced the association of CtBP with the p16 promoter and diminished p16 expression. A, CtBP was physically associated with the p16 promoter in human fibroblasts. Top, agarose gel of p16-promoter PCR products after ChIP with anti-CtBP antibody or control rabbit IgG. Prior CtBP knockdown (CtBP-kd) diminished the ChIP signal. Bottom, quantitative PCR of regions near the p16 transcription start site in anti-CtBP ChIP. Positions of centers of amplified products relative to the p16 translation start site; transcription start of p16 reference sequence NM_000077.3 is –212. B, 5 d of hypoxia increased association of CtBP with the p16 promoter (top, quantitative ChIP) and decreased p16 mRNA levels (bottom, quantitative PCR of cDNA; columns, mean ratio to normoxic levels; bars, SE) in human fibroblasts. C, CtBP knockdown blocked hypoxic repression of p16 expression. Western blots of extracts taken from human fibroblasts grown for 2 wk in hypoxia (+) or in normal atmospheric oxygen (–). D, a diagram of integrated control of p16 expression via release of CtBP-mediated repression. The indicated stresses are associated with increased p16 expression (1, 7) and with loss of CtBP-mediated repression (5, 6). CtBP does not bind directly to DNA; its binding partner(s) at the p16 promoter are not yet known.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Our findings—that CtBP mediates the repression of p16, that loss of this repression resolves the epigenetic state of the p16 promoter into an activated form and increases p16 expression, and that ambient oxygen can affect p16 expression via CtBP—clarify several aspects of the regulation of this tumor suppressor.

First, CtBP-mediated repression helps explain how a variety of stresses and stimuli can increase p16 expression (Fig. 4D) and thus provide selective pressure for loss of the p16/pocket-protein pathway in tumors. As noted in the Introduction, signals already known to relieve CtBP-mediated repression include those due to oxidative stress, UV exposure, and wound healing. Any new mechanisms identified as regulating CtBP will now also be candidates for regulating p16 expression.

Second, CtBP-mediated repression provides a previously unknown differential regulation of the two tumor-suppressor products of the human CDKN2A gene, p16 and p14ARF. In primary human cells, CtBP more specifically controls p16, the gene product more highly associated with human tumor suppression (1).

Third, CtBP-mediated repression provides a way to affect Polycomb-based repression of p16 (3, 4). No upstream molecular target that could couple cellular signals to the release of this repression and to increased p16 expression had previously been identified.

Fourth, CtBP-mediated repression might help explain the increase in p16 attributed to culture stress (2). During the passage of primary human cells in vitro, the H3K27me3 epigenetic mark at the p16 promoter is lost and p16 levels increase (3). Our findings that CtBP knockdown diminishes this mark, increases p16 expression, and accelerates senescence make CtBP a candidate for mediating the effects of culture stress; in particular, the effects of oxygen on CtBP-mediated repression (Fig. 4B and C) could mediate stress from atmospheric oxygen (14). This mechanism might also help explain the long-appreciated excess of p16 defects in tumor cell lines versus their primary tumors of origin (15). Any hypoxic repression of p16 via CtBP in primary tumors would be diminished in transfer to standard tissue culture conditions, providing selection for cells that lose p16.

Fifth, our results suggest two novel ways that hypoxia in solid tumors can repress the p16 tumor suppressor. Hypoxia represses p16 expression via CtBP (Fig. 4B and C). Also, loss of CtBP-mediated repression leads to loss of the H3K27me3 epigenetic mark at the p16 promoter (Fig. 3C). Silencing of genes through DNA methylation is favored at promoters that bear this mark, particularly at bivalently marked promoters like p16 (12, 16). Continued maintenance by hypoxia of CtBP-mediated repression and this epigenetic mark thus could favor silencing of p16 by DNA methylation.

Our results clearly show that CtBP regulates p16 expression and senescence. Nevertheless, senescence can occur through other pathways; and there may be effects of CtBP on senescence other than via p16. For example, the p400 protein that interacts with amino acids 26 to 35 of E1A regulates senescence via p53 and p21 rather than via p16 (17). We found no potential effects of CtBP knockdown on p21 (data not shown), but we cannot exclude contributions of mechanisms besides increased p16 to the diminished cell proliferation and accelerated senescence after CtBP knockdown. In particular, the interactions of CtBP with components of Polycomb complexes (6) suggest that CtBP regulates additional Polycomb-repressed genes, some of which may affect proliferation and senescence. We also cannot rule out influences in addition to CtBP on p16 expression in hypoxia (18), although the effects of hypoxia both on association of CtBP with the p16 promoter and on p16 expression, together with the lack of effect of hypoxia on p16 expression after CtBP knockdown, provide strong support for a major role for CtBP.

Our identification of CtBP as an upstream regulator of p16 expression, the wide variety of mechanisms that can affect CtBP-mediated repression, and the influence of CtBP on epigenetic regulation of p16 all suggest that CtBP may be a final common pathway for regulating p16 expression. Thus, a significant part of the question of how this tumor suppressor pathway is regulated now becomes a question of how CtBP-mediated repression is regulated.


   Disclosure of Potential Conflicts of Interest
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 Abstract
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 Materials and Methods
 Results
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 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Flight Attendant Medical Research Institute, Norman Knight Fund for Head and Neck Cancer, and Murphy Cancer Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Nick Dyson, James Koh, Scott Lowe, Yang Shi, and G. Chinnadurai for gifts of reagents; Nick Dyson and Leif Ellisen for advice and comments; Jay Kwon for technical assistance; and Takafumi Katayama for help with early experiments.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Current address for A.H. Baird: Yale School of Medicine, New Haven, CT 06510.

Received 4/ 7/08. Revised 5/20/08. Accepted 6/ 4/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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  4. Kotake Y, Cao R, Viatour P, et al. pRB family proteins are required for H3K27 trimethylation and polycomb repression complexes binding to and silencing p16INK4a tumor suppressor gene. Genes Dev 2007;21:49–54.[Abstract/Free Full Text]
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  8. Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol 2006;82:699–757.[CrossRef][Medline]
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  13. Mikkelsen TS, Ku M, Jaffe DB, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 2007;448:553–60.[CrossRef][Medline]
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