The official journal of INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH
PIGMENT CELL & MELANOMA Research p16INK4a deficiency promotes DNA hyper-replication and genetic instability in melanocytes Carina Fung, Gulietta M. Pupo, Richard A. Scolyer, Richard F. Kefford and Helen Rizos
DOI: 10.1111/pcmr.12062 Volume 26, Issue 2, Pages 236–246 If you wish to order reprints of this article, please see the guidelines here Supporting Information for this article is freely available here EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr
Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org
To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org
ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 26; 236–246
p16INK4a deficiency promotes DNA hyper-replication and genetic instability in melanocytes Carina Fung1, Gulietta M. Pupo1, Richard A. Scolyer2,3,4, Richard F. Kefford1,2 and Helen Rizos1, 1 Westmead Institute for Cancer Research, The University of Sydney at Westmead Millennium Institute, Westmead Hospital, Westmead, NSW, Australia 2 Melanoma Institute Australia, Sydney, NSW, Australia 3 Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, Sydney, NSW, Australia 4 Disciplines of Pathology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
KEYWORDS p16INK4a/melanoma/DNA damage/p53 PUBLICATION DATA Received 28 September 2012, revised and accepted for publication 15 December 2012, published online 29 December 2012
CORRESPONDENCE Helen Rizos, e-mail:
[email protected] doi: 10.1111/pcmr.12062
Summary Activated oncogenes restrict cell proliferation and transformation by triggering a DNA damage-dependent senescence checkpoint in response to DNA hyper-replication. Here, we show that loss of the p16INK4a cyclindependent kinase inhibitor and melanoma tumour suppressor facilitates a DNA damage response after a hyperreplicative phase in human melanocytes. Unlike cells expressing activated oncogenes, however, melanocytes depleted for p16INK4a display enhanced proliferation and an extended replicative lifespan in the presence of replication-associated DNA damage. Analysis of human benign naevi confirmed that DNA damage and loss of p16INK4a expression co-segregate closely. Thus, we propose that loss of p16INK4a facilitates tumourigenesis by promoting the proliferation of genetically unstable cells.
Introduction Genetic alterations affecting the p16INK4a tumour suppressor sequence are among the most frequent events in human cancer. Specific inactivation of p16INK4a via small deletions, somatic mutations or promoter hypermethylation has been reported in most human tumours (Forbes et al., 2006) and is the most common genetic alteration in human melanoma (Curtin et al., 2005). More than 60 distinct germline p16INK4a mutations have been identified in over 190 melanoma-prone kindreds worldwide (Goldstein et al., 2006). The tumour suppressor activity of p16INK4a presumably reflects its ability to prevent cell cycle progression in response to oncogenic stress. In particular, p16INK4a binds to and inhibits the kinase
activities of the cyclin D-dependent kinases, CDK4 and CDK6 to maintain the retinoblastoma protein, pRb in its hypophosphorylated, antiproliferative state (Serrano et al., 1993). The expression profile of p16INK4a also underscores its critical role as a tumour suppressor; p16INK4a accumulates progressively as cells near replicative senescence (Alcorta et al., 1996; Brenner et al., 1998) and in response to oncogenic signals (Serrano et al., 1997). Although the functions of p16INK4a are well recognized, the impact of p16INK4a inactivation on cellular lifespan and proliferation remain ambiguous, and the susceptibility of patients with germline inactivation of p16INK4a is not well understood. For instance, adult melanocytes and fibroblasts cultured from melanoma patients carrying inactive
Significance p16INK4a inactivation is associated with melanoma susceptibility and occurs frequently in melanoma tumours. However, the precise impact of p16INK4a loss on the behaviour of melanocytes remains unclear. We applied an RNA interference strategy and found that p16INK4a is critical for maintaining the proliferative capacity and genome integrity of human melanocytes. The depletion of p16INK4a enabled extended cell proliferation and DNA hyper-replication in the presence of a DNA damage response. p16INK4a loss also correlated with accumulation of DNA damage markers in human naevus cells. Thus, p16INK4a inactivation may facilitate melanomagenesis by enabling the extended proliferation and selection of genetically damaged melanocytes.
236
ª 2012 John Wiley & Sons A/S
p16INK4a loss triggers genetic instability
p16INK4a alleles displayed markedly varied lifespans in culture that were either comparable with p16INK4a-intact control cells or significantly extended (Brookes et al., 2004; Huot et al., 2002; Jones et al., 2007; Sviderskaya et al., 2003). Similarly, loss of p16INK4a function did not consistently enhance the proliferation of primary human cells, and p16INK4a-deficiency led to increased expression of p53 and its downstream transcriptional target p21Waf1 in melanocytes but not in fibroblasts (Bond et al., 2004; Sviderskaya et al., 2003; Voorhoeve and Agami, 2003). Likewise, whereas p16INK4a deficiency has been shown to overcome oncogene-induced senescence (Bond et al., 2004; Brookes et al., 2002; Huot et al., 2002; Jones et al., 2007), many other studies have shown that p16INK4a is not required for oncogene-driven senescence (Denoyelle et al., 2006; Haferkamp et al., 2009; Michaloglou et al., 2005; Voorhoeve and Agami, 2003). In this report, we applied a p16INK4a RNA interference strategy to examine the precise impact of p16INK4a loss in primary human melanocytes. Our results confirm that p16INK4a deficiency enhances DNA replication, proliferation and extends the replicative lifespan of these cells. The hyper-replication induced by p16INK4a depletion was CDK4/6 dependent and shared many features associated with acute oncogenic signalling. In particular, the loss of p16INK4a expression promoted the accumulation of replication-associated single-stranded DNA and activated the DNA damage checkpoint pathway (Bartkova et al., 2006; Di Micco et al., 2006). Strikingly, p53 activity was not sufficient to arrest p16INK4a-depleted melanocytes, and co-depletion of p53 did not alter the proliferation of these cells. We also show that in benign naevus cells, loss of p16INK4a expression correlated with accumulation of DNA damage markers. Thus, p16INK4a activity is critical in maintaining the replicative capacity and genome integrity of human melanocytes, and the loss of p16INK4a may facilitate melanomagenesis by enabling the extended proliferation and selection of genetically unstable melanocytes.
Results p16INK4a deficiency promotes melanocyte proliferation p16INK4a was depleted in human primary melanocytes by stably transducing highly specific silencing molecules that coexpressed Copepod GFP (copGFP) or puromycin resistance. Two independent silencing molecules targeting p16INK4a were generated (Figure S1), and the results of p16INK4a shRNA molecule #2 are depicted in detail. These p16INK4a silencing molecules target the p16INK4a-specific exon 1a and do not suppress expression of the syntenic p14ARF gene (Figure S2). All experiments included a negative control shRNA molecule without homology to any human gene. Diminished p16INK4a expression was detected 2 days post-transduction with p16INK4a-specific shRNA moleª 2012 John Wiley & Sons A/S
cules, and transduced melanocytes were analysed from this early time point (Figure 1A). As expected, p16INK4a deficiency was associated with a sustained increase in the total and proliferative hyper-phosphorylated form of the retinoblastoma protein, pRb (p-pRbS807/811) from 2 days post-transduction when compared to controltransduced cells (Figure 1A). These changes in pRb expression coincided with a rapid and significant increase in the proportion of melanocytes positive for the proliferation markers Ki67 and BrdU (Figure 1B,C). p16INK4a-depleted melanocytes proliferated faster and for an extended period of time compared with controltransduced melanocytes (Figure 1D). These cultured human melanocytes eventually ceased proliferating and expressed several markers of replicative senescence, including the appearance of flattened and enlarged cells and increased SA-ß galactosidase activity (data not shown). Similarly, the depletion of p16INK4a did not prevent the induction of oncogene-induced senescence and the expression of oncogenic BRAFV600E in p16INK4a-deficient melanocytes efficiently blocked cellular proliferation and induced senescence-associated heterochromatin foci as early as 3 days post-infection (Figure S3). Depletion of p16INK4a promotes DNA hyper-replication The early transcriptional impact of p16INK4a loss was also tested using microarray analysis, and the RNA expression profile of p16INK4a-deficient melanocytes relative to control cells is depicted in Table S1. Noteworthy among the set of 169 genes that were differentially expressed (P-value < 0.01 from triplicate silencing experiments), transcriptional targets of E2F were highly enriched (P-value for E2F1 targets 5.3e-16), and the depletion of p16INK4a induced the transcript expression and protein accumulation of many E2F target genes involved in DNA replication, including CDC25A, cyclin A (CCNA2), cyclin B (CCNB2), CHK1, CDT1, MCM7, E2F2 and E2F7 (Table S1, Figure 2A) (Bracken et al., 2004). Given the significant influence of p16INK4a on DNA replication, we analysed the specific action of p16INK4a on the replication machinery. Replication initiation requires assembly of the hexameric mini-chromosome maintenance (MCM) helicase (MCMs 2–7) onto the origin recognition complex via a recruitment process involving the CDC6 and CDT1 assembly factors. As cells progress into S-phase, this prereplication complex matures to facilitate the recruitment of additional factors including DNA polymerases, PCNA, cyclin D1 and CDK4 (Diffley and Labib, 2002; Gladden and Diehl, 2003). To analyse replication complex assembly, we isolated chromatinbound, actively engaged replication proteins. As shown in Figure 2B, depletion of p16INK4a significantly increased the chromatin-associated pool of PCNA, MCM7 and CDK4 (Figure 2B). 237
Fung et al. A
B
C
D
p16INK4a deficiency activates the DNA damage response The gene expression array data also indicated that many genes involved in the DNA damage response (12/ 169; P-value < 1e 6) were upregulated upon p16INK4a depletion (Table S1). We confirmed that p16INK4a depletion promoted the accumulation of phosphorylated forms of CHK1, CHK2 and p53 and increased the expression of the p53-downstream target and CDK inhibitor, p21Waf1 (Figure 3A). Examination of DAPI238
Figure 1. DNA replication is increased in melanocytes depleted for p16INK4a. (A) Human melanocytes were transduced with p16INK4a shRNA #2 or control shRNA and the expression levels of the indicated proteins were compared with those observed in untransduced melanocytes. Transduced cells were analysed several days post-transduction as indicated. (B) Cell proliferation (Ki67) was analysed and quantified 5 days after infection of melanocytes with control, p16INK4a #1 and p16INK4a #2 shRNAs. Percentage of cells positive for Ki67 is shown in histograms, which correspond to the mean SD of at least two independent transduction experiments from a total of at least 400 cells. Paired t-test was used to determine statistical significance. (C) BrdU incorporation was analysed and quantified 5 days after infection as detailed above. Paired t-test was used to determine statistical significance. (D) Growth rate and lifespan of control and p16INK4a-depleted melanocytes. Efficacy of p16INK4a silencing was confirmed by western blot analysis at days 5 (shown), 117, 132, 166 and 221 days post-transduction.
stained melanocytes by indirect immunofluorescence also revealed that melanocytes expressing p16INK4asilencing molecules had clearly detectable signs of a DNA damage response, including the accumulation of DNA damage foci marked by H2AX phosphorylation (cH2AX) and expression of the p53 binding protein 1 (53BP1) and the mediator of DNA damage checkpoint protein 1 (MDC1) (Figure 3B). Analysis of p16INK4a-depleted melanocytes showed that many cells with evidence of DNA damage frequently incorporated BrdU (Figure 3C) suggesting that DNA ª 2012 John Wiley & Sons A/S
p16INK4a loss triggers genetic instability
A
B
Figure 2. Depletion of p16INK4a increases expression of DNA replication proteins and assembly of prereplication complexes. (A) Expression of the indicated proteins was determined by western blot analysis 5 days after infection of melanocytes with lentiviruses expressing control or p16INK4a #2 shRNA molecules. (B) Control and p16INK4a-depleted melanocytes were subjected to chromatin fractionation to monitor the association of MCM7, PCNA and CDK4 with chromatin. The presence of methylated histone H3 (H3K9Me) and absence of a-tubulin were used to assess the purity of the chromatin fractions.
damage is associated with ongoing DNA replication. Further, because p16INK4a loss deregulated replication protein expression and assembly, we looked for evidence of DNA replication stress in p16INK4a-depleted melanocytes. Consistent with replication stress, melanocytes lacking p16INK4a accumulated in S and G2 phases of the cell cycle (Figure 3D), showed evidence of singlestranded DNA (i.e. BrdU immunofluorescence staining under non-denaturing conditions; Figure 3B), which is typically associated with stalled DNA replication forks and accumulated increased amounts of foci containing the single-stranded DNA-binding replication protein A (RPA) (Figure 3B). Finally, p16INK4a-depleted melanocytes expressed increased transcript levels and activation of the replication checkpoint effector kinase CHK1 and weaker activation of the complementary CHK2 kinase that responds primarily to double-stranded DNA breaks (Figure 3A) (Lambert and Carr, 2005). ª 2012 John Wiley & Sons A/S
DNA hyper-replication in p16INK4a-null melanocytes involves the CDK4/6-induced inactivation of pRb To investigate the specific role of the p16INK4a target kinases, CDK4 and CDK6 in promoting DNA hyperreplication and DNA damage on p16INK4a depletion, we introduced the melanoma-associated CDK4R24C mutant into primary melanocytes. This mutation has been identified in the germline of patients predisposed to melanoma and renders CDK4 insensitive to p16INK4a inhibition (Wolfel et al., 1995; Zuo et al., 1996). As shown in Figure 4A, melanocytes expressing CDK4R24C behaved as their p16INK4a-depleted counterparts. In particular, these melanocytes proliferated faster, showed increased BrdU incorporation, displayed abundant DNA damage foci (marked by c-H2AX) and evidence of an activated p53DNA damage pathway (p-CHK2, p-p53 and p21Waf1 accumulation). We then inhibited the activity of CDK4/6 using the highly selective small molecule inhibitor, PD0332991 (Fry et al., 2004). The addition of PD0332991, 24 h postp16INK4a silencing, completely inhibited the DNA replication and DNA damage response associated with p16INK4asuppression in human melanocytes (Figure 4B). Sustained depletion of p16INK4a permits rapid melanocyte proliferation in the presence of a p53 response We have shown that the loss of p16INK4a expression promoted a DNA damage response and activation of p53, but failed to induce sustained cell cycle arrest. To confirm the contribution of the DNA damage response to p53 induction, we suppressed ATM and ATR kinase activity with the addition of 4 mM caffeine for 3 days. As expected, the addition of caffeine inhibited phosphorylation of the ATM targets, CHK2 and p53 (Figure 5A). The phosphorylation and activation of p53 was also potently suppressed by the addition of caffeine in p16INK4adepleted melanocytes confirming that p16INK4a loss promoted an ATM/ATR-dependent DNA damage response that induced p53 activity. Further, the depletion of p53 did not significantly enhance the proliferation of control or p16INK4a-depleted melanocytes (Figure 5B) confirming that p53 does not regulate the proliferative capacity of early passage melanocytes lacking p16INK4a. Finally, we examined whether cultured melanocytes were susceptible to p53-mediated cell cycle arrest. We treated cultured melanocytes with nutlin-3, a small molecule inhibitor of the hdm2-p53 interaction, which promotes the non-genotoxic stabilization of p53 (Vassilev et al., 2004). Nutlin-3 (10 lM) induced the accumulation of p53 and promoted rapid cell cycle arrest in both control-transduced and p16INK4a-depleted melanocytes. Notably, levels of p53 and its downstream target p21Waf1, in melanocytes exposed to nultin-3, were significantly higher than seen in p16INK4a-depleted melanocytes (Figure 5C).
239
Fung et al.
A
B
C
D
Figure 3. DNA damage accumulation and checkpoint response in p16INK4a-depleted melanocytes. (A) Expression of the indicated proteins was determined by western blot analysis 5 days after infection of melanocytes with lentiviruses expressing control or p16INK4a #2 shRNA molecules. (B) Foci of DNA damage response proteins (53BP1, c-H2AX, MDC1 and RPA32) in control-transduced and p16INK4a-depleted melanocytes, 2 days post-transduction. The presence of single-stranded DNA was visualized by indirect BrdU immunofluorescence staining under non-denaturing conditions (Raderschall et al., 1999). Mean percentage SD indicate the fraction of foci-positive cells 2 days post-transduction. (C) BrdU incorporation and c-H2AX staining were examined in control-transduced and p16INK4a-depleted melanocytes 2 days post-transduction. Mean percentage SD indicate the fraction of foci-positive cells 2 days post-transduction. (D) Cell cycle analysis of control-transduced and p16INK4adepleted melanocytes 5 days post-transduction.
Loss of p16INK4a correlates with DNA damage in melanocyte tumours To test whether loss of p16INK4a might influence genetic instability in human benign melanocytic tumours, we assessed the expression of p16INK4a and c-H2AX in a panel of formalin-fixed, paraffin-embedded human benign naevi. We screened a panel of twenty benign naevi and analysed nine specimens that displayed detectable cH2AX foci and heterogeneous p16INK4a staining within the 240
naevus. This allowed for the direct comparison of neighbouring p16INK4a-positive and -negative cells within individual naevi. Using dual immunofluorescence, we showed that lack of p16INK4a expression was strongly associated with increased amounts of DNA damage as assessed by c-H2AX-positive foci in human naevus cells. First, we noted that the proportion of c-H2AX-positive foci in p16INK4a-positive cells was strongly correlated with that in p16INK4a-negative cells, that is, these lesions varied in their load of DNA damage, and this variation was ª 2012 John Wiley & Sons A/S
p16INK4a loss triggers genetic instability
A
minus that in p16INK4a-positive naevus cells was 20.2% (95% CI 13.5–27.0; P < 0.001) (Figure 6). We also confirmed that these naevi were negative for Ki67 staining and rarely expressed p53 (data not shown). Thus, naevus cells lacking p16INK4a did not proliferate nor show consistent p53 accumulation but were associated with increased DNA damage.
Discussion
B
Figure 4. CDK4/6 activity is required for DNA hyper-replication in melanocytes depleted for p16INK4a expression. (A) Melanocytes were transduced with the CDK4R24C mutant or control empty vector. Five days post-transduction the expression of the indicated proteins was analysed. BrdU incorporation and the percentage of c-H2AX foci were quantified 5 days after infection and histograms correspond to the mean SD of at least two independent transduction experiments from a total of at least 400 cells. (B) Melanocytes depleted for p16INK4a were treated with the 1 lM of the CDK4 inhibitor (CDK4i) PD0332991, 24 and 96 h post-transduction. Five days posttransduction the expression of the indicated proteins was analysed. BrdU incorporation and the percentage of c-H2AX foci were analysed and histograms correspond to the mean SD of at least two independent transduction experiments from a total of at least 400 cells.
reflected in both subpopulations. However, in each of the nine informative lesions, DNA damage levels were higher in the p16INK4a-negative cells than in the p16INK4a-positive cells: the average difference in the proportion of naevus cells displaying c-H2AX-positive foci in p16INK4a-negative ª 2012 John Wiley & Sons A/S
The DNA replication checkpoint imposes an important barrier to tumourigenesis by maintaining genome stability under conditions of replicative stress, a state of inefficient DNA replication. The activation of this checkpoint by aberrant oncogenic activity promotes senescence in nontransformed cells (Bartkova et al., 2006; Di Micco et al., 2006; Gorgoulis et al., 2005; Halazonetis et al., 2008; Vafa et al., 2002) and markers of replication stress (cH2AX and p-CHK2) and senescence co-segregate in human precancerous lesions of the lung, breast, skin, colon and bladder (Bartkova et al., 2005; Gorgoulis et al., 2005). In this study, we show that depletion of the CDK inhibitor p16INK4a in human melanocytes also induces the activation of a DNA damage checkpoint that is associated with DNA replication stress. Our results confirm a strong association between loss of p16INK4a expression and DNA damage in human benign naevi. Intriguingly, this DNA damage checkpoint was not sufficient for the initiation of melanocyte arrest in vitro, as p16INK4a-depleted melanocytes continued to proliferate in the presence of genetic instability. In contrast, human naevi were growth arrested irrespective of their p16INK4a expression status and in the absence of detectable p53 and it is likely that factors independent of p16INK4a and p53 drive naevus arrest. Our data indicate that the enhanced genetic plasticity resulting from the inactivation of p16INK4a facilitates the initiation and progression of melanoma. While p16INK4a deficiency has been associated with lifespan extension in human melanocytes (Sviderskaya et al., 2003), to our knowledge, no previous study has demonstrated the association between markers of genetic instability in human melanocytes and naevi. The mechanism by which p16INK4a inactivation promotes genetic instability reflects its CDK4/6 inhibitory activity. Activation of CDK4/6 kinases regulates DNA replication by enhancing the expression of the CDC6 and CDT1 recruitment proteins and promoting the assembly of a competent prereplication complex (Braden et al., 2008; Gladden and Diehl, 2003). Consequently, the constitutive activation of CDKs can influence genomic integrity (Cerqueira et al., 2009; Mailand and Diffley, 2005; Planas-Silva and Weinberg, 1997), and many oncogenes induce replication stress by enhancing the activities of CDKs (Bartkova et al., 2006; Halazonetis et al., 2008 Blomberg, 1999 #5120; Spruck et al., 1999). Similarly, loss of the CDK inhibitors WEE1 and CHK1 also promoted the accumulation of genetic damage during 241
Fung et al.
A
B
C
DNA replication (Beck et al., 2010). Our results suggest that the loss of p16INK4a triggers DNA damage during hyper-replication by deregulating the activity of CDK4/6 complexes. The impact of p16INK4a depletion would be more pronounced in melanocytes because p16INK4a acts as the dominant regulator of cell cycle progression and senescence in this cell type (Sviderskaya et al., 2003). The diminished checkpoint control of p16INK4a-depleted melanocytes requires detailed investigation, but may reflect the low intensity of DNA damage checkpoint signalling. In particular, the activation of p53 was modest when compared to Nutlin-3 treatment, and although the ATR-CHK1 module was activated, the downstream degradation of CDC25A, a critical component of CHK1-mediated proliferative arrest (reviewed in Lam and Rosen, 2004), was not evident. In line with these data, low levels of RAS activation also failed to induce p53 and arrest mammary epithelial cells in a transgenic mouse model, whereas high levels of RAS activation promoted p53 accumulation and cell senescence (Sarkisian et al., 2007). Similarly, loss of PTEN induced replication stress that was associated with increased proliferation because PTEN-null cells mounted an attenuated CHK1 and p53 response (Kishimoto et al., 2003; Mayo et al., 2002; Puc et al., 2005). The p53 pathway also appears to play a subordinate role in 242
Figure 5. p53 does not regulate proliferation of young p16INK4a-depleted melanocytes. (A) Melanocytes transduced with lentivirus expressing control or p16INK4a #2 shRNA molecules and cultured for 3 days in the presence (+) or absence ( ) of 4 mM caffeine. Expression of the indicated proteins was determined by western blot analysis 8 days after infection. (B) Western blot analyses (5 days posttransduction) and growth rate of control, p16INK4a-depleted, p53-depleted and p16INK4a/p53-depleted melanocytes. (C) Melanocytes transduced with lentivirus expressing control or p16INK4a #2 shRNA molecules and cultured for 24 h with 10 lM nutlin-3. (Upper panel) Expression of the indicated proteins was determined by western blot analysis 5 days after infection. (Lower panel) The percentage of BrdU positive melanocytes treated with DMSO or nutlin-3.
melanocyte homoeostasis. Melanocytes undergo replicative senescence without the progressive accumulation of p53 or p21Waf1 (Sviderskaya et al., 2003), and p53 expression is not associated with arrested naevi in vivo (Michaloglou et al., 2005; Tran et al., 2012). Finally, whereas the co-depletion of p16INK4a and p53 provided a growth advantage in fibroblasts (Voorhoeve and Agami, 2003), we show that p53 loss did not enhance the growth of early passage p16INK4a-null melanocytes. Our results provide evidence that melanocytes lacking p16INK4a are genetically unstable and prone to acquiring additional mutations that may cooperate to facilitate tumour growth. Although additional mechanisms of checkpoint control clearly initiate and maintain cell cycle arrest in p16INK4a-null melanocytes both in vitro and in vivo (Haferkamp et al., 2009; Sviderskaya et al., 2003; Tran et al., 2012), these mechanisms may be more susceptible to damage in a p16INK4a-depleted environment. This is in line with data showing that p16INK4adeficient melanocytes derived from melanoma-prone individuals are susceptible to hTERT-mediated immortalisation during which they accumulate chromosomal changes (Sviderskaya et al., 2003). Finally, the loss of p16INK4a does not promote the proliferation (Voorhoeve and Agami, 2004) nor engage the DNA damage ª 2012 John Wiley & Sons A/S
p16INK4a loss triggers genetic instability
A
Figure 6. Co-segregation of p16INK4a and c-H2AX in human naevi. Paraffin-embedded sections of human naevi were subjected to dual fluorescence immunohistochemistry with c-H2AX (red) and p16INK4a (green). Nuclei were stained with DAPI (blue). Scatter plot analysis showing the percentage of p16INK4a-positive cells scoring positive for c-H2AX versus the percentage of p16INK4a–negative naevus cells scoring positive for c-H2AX foci. The average difference in the proportion of naevus cells displaying c-H2AX-positive foci in p16INK4a-negative minus that in p16INK4apositive naevus cells was 20.2% (95% CI 13.5–27.0; P < 0.001).
B
machinery of human fibroblasts (Tort et al., 2006), supporting the distinct role of p16INK4a as a highly penetrant melanoma tumour suppressor.
Methods
(Haferkamp et al., 2009). Cells were treated with 4 mM caffeine (Sigma-Aldrich, St. Louis, MO, USA), 10 lM nutlin-3 or 1 lM PD0332991 (Selleck, Houston, TX, USA). To generate growth curves, cells were detached with trypsin, counted in triplicate using a hemocytometer, and replated at a recorded density. The relative population increase was calculated and converted to number of population doublings.
Specimen collection The formalin-fixed, paraffin-embedded human naevi (Table S2) were surgically excised between 1993 and 2010. Utilization of these specimens for this study was approved by the Sydney South West Area Health Service institutional ethics review committee (RPAH Zone) under Protocol No. X08-0155/HREC 08/RPAH/262 and Protocol No. X11-0023/HREC 11/RPAH/32. All naevi have undergone routine histopathology for diagnosis.
RNA extraction and microarray gene expression analysis Total RNA was extracted from 2 9 106 cells using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA) and arrayed as described previously (Scurr et al., 2010).
Lentiviral transductions Lentiviruses were produced in HEK293T cells as described previously (Haferkamp et al., 2009). Cells were infected using a multiplicity of infection between 5 and 10 to provide an efficiency of infection above 90%.
Cell cycle analysis For cell cycle analysis, cells were fixed in 70% ethanol at 4°C for at least 1 h, washed in PBS and stained with propidium iodide (50 ng/ll) containing ribonuclease A (50 ng/ll) for 20 min at 37°C. DNA content from at least 2000 cells was analysed using ModFIT software (Verity Software House, Topsham, ME, USA).
Constructs Cell culture and compounds Human neonatal epidermal melanocytes were obtained from Cell Applications (San Diego, CA) and cultured as previously described
ª 2012 John Wiley & Sons A/S
The p16INK4a shRNA #1 and #2 sequences correspond to nucleotides 288–306 and 236–250; NM_000077 (Voorhoeve and Agami, 2003). The p53 shRNA corresponds to nucleotides 956–974; NM_000564.
243
Fung et al. All shRNAs were cloned into the pSIH-H1-copGFP and pSIH-H1-Puro lentiviral vectors which express copGFP or puromycin resistance, respectively (System Biosciences, Mountain View, CA, USA). The non-silencing negative control shRNA did not show complete homology to any known human transcript and had the following sequence: 5′-TTAGAGGCGAGCAAGACTA-3′. The HA-tagged CDK4R24C cDNA was cloned into the pCDH-CMV-MCS-EF1-copGFP lentiviral vector, which co-expresses copGFP (System Biosciences).
Western blotting Total cellular proteins were extracted at 4°C using RIPA lysis buffer containing protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (Roche). Proteins (30–50 lg) and were resolved on 12% SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Western blots were probed with antibodies against p16INK4a (JC8; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21Waf1 (6B6; Becton Dickinson, Franklin Lakes, NJ, USA), p53 (DO-1; Santa Cruz), p-pRb (Cell Signalling, Danvers, MA, USA), pRb (G3-245; Becton Dickinson), pp53 (Ser15; Cell Signalling), H2AX (JBW301; Millipore, Bedford, MA), p-Chk1 (Cell Signalling), p-Chk2 (Cell Signalling), cyclin A (BF683; Becton Dickinson), cyclin B1 (GNS1; Santa Cruz), CDC6 (DCS-180; Santa Cruz), MCM7 (EP1974Y; Abcam), PCNA (24/PCNA; Becton Dickinson), CDK4 (DCS-31; Sigma-Aldrich), CDC25A (5H51; Santa Cruz), ß-actin (AC-74; Sigma-Aldrich), a-tubulin (B5-1-2; SigmaAldrich) and H3K9Me (Millipore).
Indirect immunofluorescence Cells were seeded on coverslips in 12-well plates at 3 9 104 cells per well at each time point, incubated overnight and stained as previously detailed (Haferkamp et al., 2009). Primary antibodies used were: Ki67 (MIB-1; DAKO, Glostrup, Denmark), c-H2AX (JBW301; Millipore and EP854(2)Y; Novus, Littleton, CO), MDC1 (Bethyl, Montgomery, TX, USA), 53BP1 (Ab-1; Oncogene, San Diego, CA, USA), RPA32 (RPA32-19; Calbiochem, Darmstadt, Germany). Comparative immunofluorescence analyses were performed in parallel with identical acquisition parameters. At least 400 cells were screened for each antigen from at least two independent experiments. For BrdU staining, cells were treated with 2 ll/mL Cell Proliferating Labelling Reagent (GE Healthcare, Buckinhamshire, UK) overnight. After fixing and permeabilization, cells were treated with RQ1 DNase (Promega, Madison, WI, USA) for 30 min and then incubated with anti-BrdU (BU-1; GE Healthcare) for 50 min, washed then incubated with Alexa Fluor 594-conjugated secondary IgG.
Immunohistochemistry Paraffin-embedded sections were antigen retrieved (Scurr et al., 2010) then blocked in 50% FCS/1% BSA/1% Tween 20 in TBS (0.9% NaCl, 20 mM Tris-Cl pH 7.4) for 1 h. Sections were incubated with primary antibodies for 2 h followed by washing three times in TBS containing 0.05% Tween 20. Subsequently, the slides were incubated with Alexa Fluor-594 and -488 secondary antibodies (Invitrogen) for 1 h, washed with TBS/0.05% Tween 20 and mounted using Prolong antifade with DAPI (Invitrogen). Primary antibodies used were: c-H2AX (20E3; Cell Signalling), p16INK4a (JC8; Santa Cruz) images were acquired as described above.
Chromatin binding assay Cells were lysed in cold CSK buffer I (10 mM PIPES pH 6.8, 300 mM sucrose, 100 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 1 mM DTT) containing 0.1% Triton X-100 and protease inhibitors (Roche) for 15 min on ice. Nuclei were pelleted by centrifugation at 360 g for 5 min. To release chromatin-bound proteins, the pellets were
244
re-extracted in CSK buffer II (10 mM PIPES pH 6.8, 300 mM sucrose, 50 mM NaCl, 6 mM MgCl2, 1 mM DTT) containing 4 U Turbo DNase I (Ambion Life Technologies, Carlsbad, CA, USA) at 37° C for 30 min followed by extraction with (NH4)2SO4 for 15 min at 37° C. Insoluble proteins were pelleted by centrifugation at 360 g for 5 min, and the soluble chromatin-bound proteins were collected.
Statistical analysis Paired t-tests were used to compare the proliferation of transduced melanocytes and the proportion of cells with c-H2AX-positive DNA damage foci in p16INK4a-positive and p16INK4a-negative naevus cells.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements We thank Karen Byth for statistical analysis. This work is supported by Program Grant 633004 and project grants of the National Health and Medical Research Council of Australia (NHMRC) and an infrastructure grant to Westmead Millennium Institute by the Health Department of NSW through Sydney West Area Health Service. Westmead Institute for Cancer Research is the recipient of capital grant funding from the Australian Cancer Research Foundation. HR is a recipient of a Cancer Institute New South Wales, Research Fellowship and a NHMRC Senior Research Fellowship. RAS is a Cancer Institute New South Wales Clinical Research Fellow. The support of the Melanoma Foundation of the University of Sydney and colleagues from the Melanoma Institute Australia (incorporating the Sydney Melanoma Unit) and the Department of Tissue Pathology and Diagnostic Oncology at the Royal Prince Alfred Hospital are also gratefully acknowledged.
References Alcorta, D.A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., and Barrett, J.C. (1996). Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. U S A 93, 13742–13747. Bartkova, J., Horejsi, Z., Koed, K. et al. (2005). DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870. Bartkova, J., Rezaei, N., Liontos, M. et al. (2006). Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444, 633–637. Beck, H., Nahse, V., Larsen, M.S., Groth, P., Clancy, T., Lees, M., Jorgensen, M., Helleday, T., Syljuasen, R.G., and Sorensen, C.S. (2010). Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase. J. Cell Biol. 188, 629– 638. Blomberg, I., and Hoffmann, I. (1999). Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol. Cell Biol. 19, 6183–6194. Bond, J., Jones, C., Haughton, M., Demicco, C., Kipling, D., and Wynford-Thomas, D. (2004). Direct evidence from siRNA-directed “knock down” that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation. Exp. Cell Res. 292, 151–156. Bracken, A.P., Ciro, M., Cocito, A., and Helin, K. (2004). E2F target genes: unraveling the biology. Trends Biochem. Sci. 29, 409–417. Braden, W.A., Mcclendon, A.K., and Knudsen, E.S. (2008). Cyclindependent kinase 4/6 activity is a critical determinant of prereplication complex assembly. Oncogene 27, 7083–7093.
ª 2012 John Wiley & Sons A/S
p16INK4a loss triggers genetic instability Brenner, A.J., Stampfer, M.R., and Aldaz, C.M. (1998). Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene 17, 199–205. Brookes, S., Rowe, J., Ruas, M. et al. (2002). INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence. EMBO J. 21, 2936–2945. Brookes, S., Rowe, J., Gutierrez Del Arroyo, A., Bond, J., and Peters, G. (2004). Contribution of p16(INK4a) to replicative senescence of human fibroblasts. Exp. Cell Res. 298, 549–559. Cerqueira, A., Santamaria, D., Martinez-Pastor, B., Cuadrado, M., Fernandez-Capetillo, O., and Barbacid, M. (2009). Overall Cdk activity modulates the DNA damage response in mammalian cells. J. Cell Biol. 187, 773–780. Curtin, J.A., Fridlyand, J., Kageshita, T. et al. (2005). Distinct sets of genetic alterations in melanoma. N. Engl. J. Med. 353, 2135– 2147. Denoyelle, C., Abou-Rjaily, G., Bezrookove, V. et al. (2006). Antioncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway. Nat. Cell Biol. 8, 1053–1063. Di Micco, R., Fumagalli, M., Cicalese, A. et al. (2006). Oncogeneinduced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444, 638–642. Diffley, J.F., and Labib, K. (2002). The chromosome replication cycle. J. Cell Sci. 115, 869–872. Forbes, S., Clements, J., Dawson, E. et al. (2006). COSMIC 2005. Br. J. Cancer 94, 318–322. Fry, D.W., Harvey, P.J., Keller, P.R. et al. (2004). Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol. Cancer Ther. 3, 1427–1438. Gladden, A.B., and Diehl, J.A. (2003). The cyclin D1-dependent kinase associates with the pre-replication complex and modulates RB.MCM7 binding. J. Biol. Chem. 278, 9754–9760. Goldstein, A.M., Chan, M., Harland, M. et al. (2006). High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL. Cancer Res. 66, 9818–9828. Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P. et al. (2005). Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913. Haferkamp, S., Scurr, L.L., Becker, T.M., Frausto, M., Kefford, R.F., and Rizos, H. (2009). Oncogene-induced senescence does not require the p16(INK4a) or p14ARF melanoma tumor suppressors. J. Invest. Dermatol. 129, 1983–1991. Halazonetis, T.D., Gorgoulis, V.G., and Bartek, J. (2008). An oncogene-induced DNA damage model for cancer development. Science 319, 1352–1355. Huot, T.J., Rowe, J., Harland, M. et al. (2002). Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts. Mol. Cell. Biol. 22, 8135–8143. Jones, R., Ruas, M., Gregory, F., Moulin, S., Delia, D., Manoukian, S., Rowe, J., Brookes, S., and Peters, G. (2007). A CDKN2A mutation in familial melanoma that abrogates binding of p16INK4a to CDK4 but not CDK6. Cancer Res. 67, 9134–9141. Kishimoto, H., Hamada, K., Saunders, M., Backman, S., Sasaki, T., Nakano, T., Mak, T.W., and Suzuki, A. (2003). Physiological functions of Pten in mouse tissues. Cell Struct. Funct. 28, 11–21. Lam, M.H., and Rosen, J.M. (2004). Chk1 versus Cdc25: checking one’s levels of cellular proliferation. Cell Cycle 3, 1355–1357. Lambert, S., and Carr, A.M. (2005). Checkpoint responses to replication fork barriers. Biochimie 87, 591–602. Mailand, N., and Diffley, J.F. (2005). CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/Cdependent proteolysis. Cell 122, 915–926.
ª 2012 John Wiley & Sons A/S
Mayo, L.D., Dixon, J.E., Durden, D.L., Tonks, N.K., and Donner, D.B. (2002). PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy. J. Biol. Chem. 277, 5484–5489. Michaloglou, C., Vredeveld, L.C., Soengas, M.S., Denoyelle, C., Kuilman, T., Van Der Horst, C.M., Majoor, D.M., Shay, J.W., Mooi, W.J., and Peeper, D.S. (2005). BRAFE600-associated senescencelike cell cycle arrest of human naevi. Nature 436, 720–724. Planas-Silva, M.D., and Weinberg, R.A. (1997). Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution. Mol. Cell. Biol. 17, 4059–4069. Puc, J., Keniry, M., Li, H.S. et al. (2005). Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell 7, 193–204. Raderschall, E., Golub, E.I., and Haaf, T. (1999). Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage. Proc. Natl Acad. Sci. USA. 96, 1921–1926. Sarkisian, C.J., Keister, B.A., Stairs, D.B., Boxer, R.B., Moody, S.E., and Chodosh, L.A. (2007). Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis. Nat. Cell Biol. 9, 493–505. Scurr, L.L., Pupo, G.M., Becker, T.M. et al. (2010). IGFBP7 is not required for B-RAF-induced melanocyte senescence. Cell 141, 717–727. Serrano, M., Hannon, G.J., and Beach, D. (1993). A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/ CDK4. Nature 366, 704–707. Serrano, M., Lin, A.W., Mccurrach, M.E., Beach, D., and Lowe, S. W. (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 85, 593– 602. Spruck, C.H., Won, K.A., and Reed, S.I. (1999). Deregulated cyclin E induces chromosome instability. Nature 401, 297–300. Sviderskaya, E.V., Gray-Schopfer, V.C., Hill, S.P. et al. (2003). p16/ Cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression. J. Natl Cancer Inst. 95, 723– 732. Tort, F., Bartkova, J., Sehested, M., Orntoft, T., Lukas, J., and Bartek, J. (2006). Retinoblastoma pathway defects show differential ability to activate the constitutive DNA damage response in human tumorigenesis. Cancer Res. 66, 10258–10263. Tran, S.L., Haferkamp, S., Scurr, L.L., Gowrishankar, K., Becker, T. M., Desilva, C., Thompson, J.F., Scolyer, R.A., Kefford, R.F., and Rizos, H. (2012). Absence of distinguishing senescence traits in human melanocytic nevi. J. Invest. Dermatol. 19, 126. Vafa, O., Wade, M., Kern, S., Beeche, M., Pandita, T.K., Hampton, G. M., and Wahl, G.M. (2002). c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol. Cell 9, 1031–1044. Vassilev, L.T., Vu, B.T., Graves, B. et al. (2004). In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848. Voorhoeve, P.M., and Agami, R. (2003). The tumor-suppressive functions of the human INK4A locus. Cancer Cell 4, 311–319. Voorhoeve, P.M., and Agami, R. (2004). Unraveling human tumor suppressor pathways: a tale of the INK4A locus. Cell Cycle 3, 616– 620. Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., Klehmannhieb, E., Deplaen, E., Hankeln, T., Zumbuschenfelde, K. H.M., and Beach, D. (1995). A p16(INK4a)-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281–1284. Zuo, L., Weger, J., Yang, Q., Goldstein, A.M., Tucker, M.A., Walker, G.J., Hayward, N., and Dracopoli, N.C. (1996). Germline mutations
245
Fung et al. in the p16ink4a binding domain of CDK4 in familial melanoma. Nat. Genet. 12, 97–99.
Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1. Targeted silencing of the p16INK4a tumor suppressor protein.
246
Figure S2. Specific inhibition of p16INK4a expression. Figure S3. Oncogenic BRAF induces senescence in p16INK4a-depleted melanocytes. Table S1. Expression of the CDKN2A isoforms and differentially expressed genes (P value
