Title: Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions
Abstract: Article8 May 2012Open Access Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions Anitha Suram Anitha Suram New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Jessica Kaplunov Jessica Kaplunov Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Priyanka L Patel Priyanka L Patel New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Haihe Ruan Haihe Ruan New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Aurora Cerutti Aurora Cerutti IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy Search for more papers by this author Virginia Boccardi Virginia Boccardi New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Marzia Fumagalli Marzia Fumagalli IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, ItalyCurrent address: TTFactor Srl, Milan, Italy Search for more papers by this author Raffaella Di Micco Raffaella Di Micco IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, ItalyCurrent address: Department of Pathology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Neena Mirani Neena Mirani Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Resham Lal Gurung Resham Lal Gurung Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Manoor Prakash Hande Manoor Prakash Hande Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Fabrizio d'Adda di Fagagna Fabrizio d'Adda di Fagagna IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Utz Herbig Corresponding Author Utz Herbig New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Anitha Suram Anitha Suram New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Jessica Kaplunov Jessica Kaplunov Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Priyanka L Patel Priyanka L Patel New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Haihe Ruan Haihe Ruan New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Aurora Cerutti Aurora Cerutti IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy Search for more papers by this author Virginia Boccardi Virginia Boccardi New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Marzia Fumagalli Marzia Fumagalli IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, ItalyCurrent address: TTFactor Srl, Milan, Italy Search for more papers by this author Raffaella Di Micco Raffaella Di Micco IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, ItalyCurrent address: Department of Pathology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Neena Mirani Neena Mirani Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Resham Lal Gurung Resham Lal Gurung Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Manoor Prakash Hande Manoor Prakash Hande Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Fabrizio d'Adda di Fagagna Fabrizio d'Adda di Fagagna IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Utz Herbig Corresponding Author Utz Herbig New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Author Information Anitha Suram1, Jessica Kaplunov2, Priyanka L Patel1, Haihe Ruan1, Aurora Cerutti3, Virginia Boccardi1, Marzia Fumagalli3, Raffaella Di Micco3, Neena Mirani4, Resham Lal Gurung5, Manoor Prakash Hande5, Fabrizio d'Adda di Fagagna3,6 and Utz Herbig 1,2 1New Jersey Medical School-University Hospital Cancer Center, UMDNJ-New Jersey Medical School, Newark, NJ, USA 2Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA 3IFOM Foundation—FIRC Institute of Molecular Oncology Foundation, Milan, Italy 4Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, Newark, NJ, USA 5Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 6Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy *Corresponding author. Department of Microbiology and Molecular Genetics, New Jersey Medical School-UH Cancer Center, UMDNJ, CC-G1226, 205 South Orange Avenue, Newark, NJ 07103, USA. Tel.:+1 973 972 4426; Fax:+1 973 972 1875; E-mail: [email protected] The EMBO Journal (2012)31:2839-2851https://doi.org/10.1038/emboj.2012.132 There is a Have you seen? (June 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans. Introduction Dysfunctional telomeres play a critical role in the progression of human cancer. In the context of deregulated DNA damage checkpoint signalling, telomere dysfunction can promote cancer progression by seeding the events that lead to chromosomal instability. This situation is observed in advanced cancer precursor lesions such as ductal carcinoma in situ (DCIS) (Chin et al, 2004) and colonic adenomas with high-grade dysplasia (Rudolph et al, 2001). When DNA damage checkpoint responses are intact, however, telomere dysfunction leads to cellular senescence, a permanent and stable proliferative arrest that functions as a cell intrinsic tumour suppressing mechanism in mouse model systems (Sharpless and DePinho; Cosme-Blanco et al, 2007; Feldser and Greider, 2007). Cells with dysfunctional telomeres have been detected in cancers with low mitotic activity, such as early stage B-cell chronic lymphocytic leukaemia, suggesting that telomere dysfunction also poses a barrier to cancer progression in humans (Augereau et al, 2011). However, direct evidence that telomere dysfunction-induced cellular senescence (TDIS) is an in vivo physiologic response that limits progression of human cancer is still missing. Cellular senescence is thought to limit cancer progression by preventing the proliferation of cells in early neoplastic lesions. Studies conducted using mouse model systems suggest that cellular senescence arrests tumour growth before cells become malignant and invade surrounding tissue (Collado and Serrano, 2010). Similarly, cells with senescence-like features have also been detected in benign human cancer precursor lesions, but are absent in malignant cancers, supporting the conclusions that this stable growth arrest limits cancer progression at premalignant stages. In mouse models, the tumour suppressing functions of cellular senescence can be triggered by oncogenes (Braig et al, 2005; Collado et al, 2005; Michaloglou et al, 2005), loss of growth regulatory mechanisms (Chen et al, 2005), or dysfunction of telomeres (Cosme-Blanco et al, 2007; Feldser and Greider, 2007), but the mechanisms ultimately triggering cellular senescence in human cancer precursor lesions are still incompletely understood. Entry into senescence is regulated by at least two signalling pathways: a stress-induced p16INK4a/Rb-dependent pathway and a DNA damage response (DDR) pathway that is mediated by p53 (Herbig and Sedivy, 2006). While the molecular activators of the p16INK4a/Rb pathway are largely unknown, p53 is activated primarily in response to DNA damage such as double-stranded DNA breaks (DSBs). In human cell cultures, a primary reason for senescence is because telomeres progressively shorten with every cell cycle until a critical length is reached that causes telomeres to become dysfunctional. Telomere erosion is a consequence of a variety of factors that include the inability of the replicative polymerase to completely duplicate linear DNA (also called 'end replication problem'), postreplicative processing of chromosome ends, and sporadic telomere attrition due to repair events at damaged telomeres (Lansdorp, 2005). Once telomeres become dysfunctional, they are sensed as DSBs and consequently activate the DDR/p53 senescence pathway (d'Adda di Fagagna et al, 2003; Takai et al, 2003; Herbig et al, 2004). Cellular senescence can also be induced prematurely before telomere shortening due to continuous cell proliferation becomes growth limiting. Dysregulated oncogenes, for example, cause cells to undergo oncogene-induced senescence (OIS) after a brief period of hyperproliferation. Depending on cell type, signal strength, and extracellular environment, oncogenes activate distinct and sometimes complex signalling networks that likely each contribute to various degrees to the permanent growth arrest that characterizes OIS (Courtois-Cox et al, 2008). Oncogenic signals also cause high levels of DNA replication stress, which leads to the formation of DSBs and activation of a persistent DDR (Bartkova et al, 2006; Di Micco et al, 2006). Since aberrant oncogene signalling frequently initiates cancer growth in humans (Hanahan and Weinberg, 2011), and signs of a persistent DDR are observed in several benign and malignant human neoplasms (Bartkova et al, 2005, 2007; Gorgoulis et al, 2005; Nuciforo et al, 2007), it is currently thought that the reasons for the inactive nature of human cancer precursor lesions is because cells within these lesions had undergone OIS. Here, we further characterize the causes for cellular senescence in cancer precursor lesions and provide compelling evidence that telomeres play a critical role in preventing malignant cancer progression in humans. Results Human nevi are comprised of cells that display hallmarks of TDIS Cells displaying senescence-like features such as senescence-associated β-galactosidase activity, elevated levels of p16, and signs of an activated DDR, have been detected in human nevi, suggesting that cellular senescence limits melanoma progression at premalignant stages (Gorgoulis et al, 2005; Michaloglou et al, 2005; Gray-Schopfer et al, 2006). To characterize specifically which senescence pathway was activated in cells of human nevi, we immunostained benign and dysplastic nevi (Supplementary Figure S1A) for p16 and for 53BP1, a cytological marker of DDR activation (Adams and Carpenter, 2006). In agreement with previous studies (Michaloglou et al, 2005; Gray-Schopfer et al, 2006), we detected heterogeneous p16 levels and a mosaic p16 expression pattern in cells of nevi (Supplementary Figure S1B and C). These data therefore suggest that the p16/Rb senescence pathway alone is not responsible for arresting melanocytic cells at this stage of cancer development. In contrast, the great majority (∼90%) of cells within 26 analysed nevi displayed discrete 53BP1 foci, independent of patient age, demonstrating a massive activation of a DDR in premalignant melanocytic lesions (Figure 1A and B; Supplementary Figure S1C and E). The majority of normal melanocytes in the dermal–epidermal junction of analysed samples lacked 53BP1 foci, demonstrating that 53BP1 focus formation is not an intrinsic property of melanocytes in tissue (Figure 1B; Supplementary Figure S1D and E). Interestingly, we observed an ageing-associated increase of 53BP1-positive normal melanocytes in the epidermis of these patients, suggesting that the observed heterogeneity in 53BP1-positive normal cells was attributed to patient age (Supplementary Figure S1E). Similar to normal melanocytes, cells of invasive melanoma showed infrequent and heterogeneous staining for 53BP1 foci. Vertical growth phase melanomas and melanocytic cells in deep soft tissue generally lacked signs of a DDR, while cells in radial growth phase melanoma often displayed discrete 53BP1 foci, albeit at a lower frequency compared with cells in nevi (Figure 1A and B; Supplementary Figure S1F). These observations are consistent with the reported downregulation of DDR activity in more advanced human cancers (Bartkova et al, 2005, 2007; Gorgoulis et al, 2005; Nuciforo et al, 2007). Together, our data demonstrate a substantial and consistent accumulation of DDR-positive melanocytes in nevi, but not in the epidermis or in malignant melanomas. Figure 1.Melanocytic cells of benign- and dysplastic nevi, but not cells of malignant melanoma, display hallmarks of telomere dysfunction-induced cellular senescence. (A) Tissue sections from indicated lesions were immunostained with antibodies against melanA (red) and 53BP1 (green). Insets display an enlarged section of the indicated area. (B) Quantitation of 53BP1-positive melanocytic cells in benign nevi (Bng; n=13, 3529 cells), dysplastic nevi (Dsp; n=13, 2300 cells), melanoma (Mel; n=18, 5401 cells), and in normal epidermal melanocytes adjacent to the lesion (Norm; n=12, 485 cells). Values are shown as mean±s.d.; *P<0.001. (C) Dysfunctional telomeres in nevi. Tissue sections from benign nevi were processed by immunoFISH to simultaneously detect 53BP1 (green) and telomeres (red). Enlarged versions of the numbered DNA damage foci showing colocalization with telomeres are shown in the right micrographs. Note that only one optical slice is displayed. (D) Quantitation of TIF positive cells in indicated lesions (mean±s.d.). A total of 13 benign nevi (1355 53BP1 foci), 13 dysplastic nevi (2968 53BP1 foci), and 7 melanoma (891 53BP1 foci) were counted; *P<0.001. (E) Distribution of telomere lengths based on their signal intensities (x-axis; arbitrary units). Top histogram: all telomeric signals in cells of nevi (average signal intensity 268±46). Bottom histogram: single (red bars; average signal intensity 281±46) and multiple/diffuse (blue bars; average signal intensity 275±56) telomeric signals associated with 53BP1 foci. n: number of telomeric signals analysed (F) Tissue sections from a dysplastic naevus (top), and invasive melanoma (bottom) were immunostained using antibodies against 53PB1 (red) and macroH2A (green). Arrows point to stromal cells and basal layer epidermal keratinocytes that did not display elevated macroH2A levels. Dashed line separates epidermis (bottom left) from naevus (top right). Scale bars: 25 μm. Statistical significance was calculated by one-way ANOVA followed by Tukey's post hoc test. Download figure Download PowerPoint In replicative senescent human cells, persistent DDR foci are frequently associated with short and dysfunctional telomeres (d'Adda di Fagagna et al, 2003; Herbig et al, 2004). To test whether the DDR was a consequence of telomere dysfunction, we analysed tumour tissue for the presence of telomere dysfunction-induced DNA damage foci (TIF), or colocalizations between telomeres, labelled using a telomeric peptide nucleic acid (PNA), and DDR foci as we have done previously (Herbig et al, 2004). Images were acquired at high magnification ( × 100) and in z-series throughout the entire thickness of the tissue using a fluorescence microscope equipped with an ApoTome. This generated images of consecutive 0.3–0.4 μm optical slices and therefore minimized any coincidental overlap between two distinct foci along the optical z-axis (Supplementary Figure S2A and B). Using this technique, we discovered that over 60% of all DDR foci analysed in nevi colocalized with telomeric repeats (Figure 1C; Supplementary Figure S2C). Similarly, the few remaining DDR foci in cells of melanoma were also primarily telomeric, albeit to a lesser degree compared with 53BP1 foci in nevi (Supplementary Figure S2C). In contrast, only 6% of DDR foci colocalized with centromeric foci, visualized using antibodies against centromeric proteins, supporting our conclusions that the DDR was specific to telomeres (Supplementary Figure S2D). Of note, the few colocalizations between centromeric signals and DDR foci also demonstrates that our imaging technique is sensitive enough to discriminate coincidental overlap between two random foci within a cell nucleus. Dysfunctional telomeres in cells of nevi retained the shelterin component TRF2, arguing against the possibility that telomere dysfunction in these cells was a consequence of TRF2 loss and/or degradation (Supplementary Figure S2E). In order to quantitate the percentages of cells containing dysfunctional telomeres in tissue, we scored cells as TIF positive when 50% of 53BP1 foci in a cell nucleus colocalized with telomeric repeats. We discovered that the majority of melanocytic cells in benign (64%) and dysplastic nevi (70%) were indeed TIF positive, which was in contrast to cells in malignant melanoma that infrequently scored as TIF positive (11%; Figure 1D). Thus, despite the apparent lack of total telomere shortening in melanocytic cells of human nevi (Michaloglou et al, 2005), the data presented here suggest that cells within these lesions had undergone TDIS. Although it has been reported that total telomere lengths in cells of nevi are similar to those of surrounding stromal cells, it is possible that the DDR in melanocytic cells was initiated by one or few telomeres that had become critically short and dysfunctional due to stochastic telomere attrition events (Michaloglou et al, 2005). To test this possibility, we quantified fluorescence signal intensities of telomeres in TIF (Figure 1E, red and blue bars) and compared these with signal intensities of telomeres not associated with DDR foci (Figure 1E, black bars). Surprisingly, our analysis revealed that telomeric foci colocalizing with 53BP1 (Figure 1E, red bars) emitted on average similar fluorescence signal intensities compared with the other telomeres in the same cells (Figure 1E, black bars), suggesting that telomere dysfunction in these cells was not primarily due to critical telomere attrition. We also discovered that ∼30% of all dysfunctional telomeres analysed displayed telomeric doublets or diffuse telomere signals, suggesting that these structures were either fragile telomeres (Sfeir et al, 2009) or alternatively, aggregation of multiple dysfunctional telomeres within one DDR focus as described recently in senescent human fibroblast cultures (Figure 1C and E; Supplementary Figure S2F) (Kaul et al, 2011). To distinguish between these two possibilities, we measured the combined fluorescence intensities of telomeric doublets in DDR foci and discovered that these combined values (Figure 1C, blue bars) were similar to intensity values of individual telomere signals in DDR foci. Given that DDR-associated telomeric aggregates almost always display greater combined fluorescence signal intensities compared with the other telomeres in the same cells (Kaul et al, 2011), it is unlikely that these aberrant telomeric structures are due to aggregation of multiple dysfunctional telomeres in a single DDR focus. Elevated levels of some heterochromatin proteins are a feature of senescent melanocytes in culture (Michaloglou et al, 2005; Bandyopadhyay et al, 2007) and have been used as a marker to detect senescent cells in tissue (Collado et al, 2005; Herbig et al, 2006; Jeyapalan et al, 2007; Majumder et al, 2008). Using antibodies against macroH2A, a late-appearing molecular component of senescence-associated heterochromatin (Zhang et al, 2005) and a suppressor of melanoma progression (Kapoor et al, 2010), we observed that virtually all melanocytic cells in analysed nevi displayed elevated macroH2A levels (Figure 1F). Similar observations have also been reported recently (Kapoor et al, 2010). Together with studies demonstrating senescence-associated β-galactosidase activity in nevi (Michaloglou et al, 2005; Gray-Schopfer et al, 2006), our observations not only support previous conclusions that cells in nevi are senescent, but also strongly suggest that elevated levels of macroH2A are a feature specific to senescent cells in vivo. In support of this, we show that nuclear staining of the cell proliferation marker Ki67 and of macroH2A was mutually exclusive in early invasive melanocytic lesions that display features of both senescence and proliferation (Supplementary Figure S3A). In contrast to cells of nevi, few normal epidermal melanocytes displayed elevated macroH2A levels (Supplementary Figure S3B). Similarly, macroH2A levels in cells of melanomas in deep soft tissue were generally low, suggesting that cells of these invasive cancers had either lost the ability to undergo senescence or did not encounter signals leading to cellular senescence (Figure 1F). In nevi, cells that stained positive for 53BP1 also displayed elevated levels of macroH2A, revealing a positive correlation between DDR activation and the senescence status (Figure 1F). No correlation between telomere dysfunction and p16 upregulation could be established (Supplementary Figure S1C). Breast- and colon-cancer precursor lesions display hallmarks of TDIS Our data are consistent with the idea that TDIS limits melanoma progression at premalignant stages. To determine whether the potential tumour suppressing functions of TDIS are limited to melanocytic cells of nevi or also affect the growth of epithelial tumour cells, we analysed colonic (tubular) adenomas (CA) and usual/atypical ductal hyperplasias (DH) of the breast for markers of TDIS (Supplementary Figure S4). Strikingly, we discovered that the great majority of epithelial cells in hyperplastic regions of CA and of DH displayed prominent 53BP1 foci (Figure 2A and B). This is in contrast to cells in normal epithelium adjacent to hyperplastic regions and cells in malignant cancers, which generally lacked 53BP1 foci (Figure 2A and B; Supplementary Figure S6A). Significantly, the great majority of DDR foci in cells of DH colocalized with telomeric repeats that were on average not shorter compared with other telomeres in these cells (Supplementary Figure S5A and B) and over 80% of cells in hyperplastic regions of the tissue were TIF positive (Figure 2C). As in premalignant melanocytic lesions, aberrant telomeric structures resembling fragile telomeres occasionally could be detected in TIF (Supplementary Figure S5B). TIF-positive cells could also be detected in CA, albeit at a lower frequency compared with DH (Supplementary Figure S5A). A likely reason is that telomeres in CA are very short, making it difficult to visualize telomeres using a PNA or antibodies against telomeric proteins (Meeker et al, 2004b) (Supplementary Figure S5C). Despite these limitations, we frequently detected dysfunctional telomeres and demonstrate a dramatic and statistically highly significant difference in TIF-positive cells between colonic adenomas and colonic carcinomas (42% versus 0.2%, respectively, P<0.001; Figure 2C). Although we did not detect aberrant telomeric structures in TIF of CA, likely due to the weak fluorescence signals emitted from telomeres, also in these premalignant lesions average lengths of telomeres in TIF were similar to the other telomeres in the cells (Supplementary Figure S5D). In contrast to cells in normal epithelium and in invasive carcinomas, 53BP1-positive cells in hyperplastic regions of CA and DH displayed elevated levels of macroH2A, providing further evidence that they were senescent (Figure 2D; Supplementary Figure S6A). As with melanocytic cells in nevi, epithelial cells that displayed high levels of macroH2A did not stain positive for the proliferation marker Ki67, corroborating our conclusions that macroH2A is a marker specific to senescent cells in tissues (Supplementary Figure S6B). Together, our data demonstrate hallmarks of TDIS in cells of precursor lesions to three distinct and common human cancers, but not in their malignant cancer counterparts. Figure 2.Epithelial cells of precursor lesions to colon- and breast-cancers display hallmarks of telomere dysfunction-induced cellular senescence. (A) Tissue sections from colonic adenomas (colon) and DH of the breast (breast; left column) and from invasive colon- and breast-carcinomas (right column) were immunostained with antibodies against 53BP1 (red, top row; green, bottom row). (B) Quantitation of 53BP1-positive epithelial cells (mean±s.d). Top graph: colonic adenomas (CA, n=10, 5588 cells), colonic adenocarcinomas (Carc, n=14, 4898 cells), epithelial cells from adjacent normal colonic mucosa (Norm, n=9, 2474 cells). Bottom graph: usual- and atypical-DH (Hyp, n=14, 4359 cells), invasive ductal carcinomas (Carc, n=15, 2905 cells), and luminal epithelial cells from adjacent normal ducts (Norm, n=9, 3153 cells); *P<0.001 by one-way ANOVA followed by Tukey's post hoc test. (C) Quantitation of TIF-positive cells (mean±s.d.). CA: n=10; 3309 foci; Carc: n=14; 205 foci; Hyp: n=14, 1510 cells; Carc: n=15, 661 cells; *P<0.001 by unpaired t-test. (D) Tissue sections from indicated lesions were immunostained using antibodies against 53PB1 (red) and macroH2A (green). Scale bar: 50 μm. Download figure Download PowerPoint Oncogenic H-RasV12 causes stalling of telomeric replication forks Although telomere erosion in human cell cultures allows ∼60 population doublings in vitro, the same number of cell divisions in vivo could potentially generate a lesion consisting of 1 × 1018 cells, a tumour mass weighing over 100 tons. We therefore reasoned that cells of precancerous lesions likely encountered stresses, which either accelerated telomere erosion or activated mechanisms that trigger telomere dysfunction. Common to all human cancer precursor lesions are stresses that lead to cellular hyperproliferation and DNA replication stress, often induced by aberrant oncogene signalling (Halazonetis et al, 2008). Since fragile sites are particularly sensitive to oncogene-induced DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006), and telomeres resemble fragile sites (Martinez et al, 2009; Sfeir et al, 2009; Ye et al, 2010), we asked whether oncogenic Ras causes DNA replication stress in telomeric repeats, thereby altering telomere structure and function. Using molecular DNA combing combined with telomeric fluorescence in situ hybridization (FISH; Figure 3A; Supplementary Figure S7A), we discovered that oncogene expression increased the fraction of DNA replication forks arresting at the transition between non-telomeric and telomeric tracts, compared with control cells (Figure 3B and C). In addition, oncogene-expressing cells accumulated partially replicated telomeres more frequently, while the numbers of fully replicated telomeres were reduced as compared with the control (Figure 3C). Indeed, the percentage of fork stalling events, defined as asymmetric DNA replication bubbles at telomeres (see Figure 3A), was dramatically increased by oncogene activation (Figure 3D). While these differences were statistically significant at telomeres (P=0.0