Review

Oncogene (2011) 30, 2087–2097; doi:10.1038/onc.2010.614; published online 7 February 2011

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

C Romagosa1, S Simonetti2, L López-Vicente1, A Mazo3, M E Lleonart1, J Castellvi1 and S Ramon y Cajal1

  1. 1Pathology Department, Fundació Institut de Recerca, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
  2. 2Pangaea Biotech, Oncology Laboratory, USP Dexeus University Institute, Barcelona, Spain
  3. 3Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain

Correspondence: Dr S Ramon y Cajal, Pathology Department, Fundació Institut de Recerca, Hospital Universitari Vall d’Hebron, Passeig de la Vall d’Hebron 119-129, Barcelona 08035, Spain. E-mail: sramon@vhebron.net

Received 11 July 2010; Revised 5 December 2010; Accepted 7 December 2010; Published online 7 February 2011.

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Abstract

p16Ink4a is a protein involved in regulation of the cell cycle. Currently, p16Ink4a is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16Ink4a has also been described in several tumors. This review attempts to elucidate when and why p16Ink4a overexpression occurs, and to suggest possible implications of p16Ink4a in the diagnosis, prognosis and treatment of cancer.

Keywords:

p16Ink4a; overexpression; cancer; senescence; high grade

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Introduction

Mammalian cells have developed complex mechanisms against mutagens and inappropriate growth stimuli as protection from malignant transformation and tumorigenesis. When irreversible tumor-producing stimuli are present, these defense systems enable cells to take roads other than proliferation, such as apoptosis or senescence (Lleonart et al., 2009). Several tumor suppressor genes involved in related pathways regulate the outcome of cell development. One of these is p16Ink4a (Alcorta et al., 1996), the focus of this review.

p16Ink4a expression has been evaluated in several tumor types with very different results, ranging from its loss or downregulation (Brambilla et al., 1999a, 1999b; Schneider-Stock et al., 2005; Ayhan et al., 2010) to its clear overexpression (Milde-Langosch et al., 2001; Armes et al., 2005; Zhao et al., 2006; Angiero et al., 2008; Buajeeb et al., 2009). The main objective of this review is to clarify these apparently contrasting expression patterns and to reveal their implications in cancer management.

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Physiological role of p16Ink4a

p16Ink4a and the cell cycle

p16Ink4a is the principal member of the Ink4 family of CDK inhibitors. It is codified by a gene localized on chromosome 9p21 within the INK4a/ARF locus, which encodes for two different proteins with different promoters: p16Ink4a and p19ARF. Both proteins have antiproliferative biological activity, and are involved in the retinoblastoma protein (Rb) and p53 pathways, respectively (Serrano, 1997; Weber et al., 2000; Pei and Xiong, 2005). These proteins and their interactions are crucial for understanding the key points of tumor suppression.

It is well known that p16Ink4a contributes to the regulation of cell cycle progression by inhibiting the S phase. The molecular pathway responsible for this inhibition is shown in Figure 1. Briefly, p16Ink4a binds to CDK4/6, inhibiting cyclin D–CDK4/6 complex formation and CDK4/6-mediated phosphorylation of Rb family members. Expression of p16Ink4a maintains the Rb family members in a hypophosphorylated state, which promotes binding to E2F1 and leads to G1 cell cycle arrest (Serrano, 1997). However, this classically known function seems to be just a simplified scheme of the global role of p16Ink4a, and many aspects of its function and regulation are still partially unresolved.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Functions and interactions of the proteins coded by the INK4a/ARF locus. Different aspects of p16Ink4a molecular pathways are explained in this figure: (i) Members of the INK4 family bind and inactivate CDK4/6, blocking phosphorylation of Rb and inducing cell cycle arrest. ARF inhibits MDM2, resulting in p53 stabilization. p53 stabilization initiates cell apoptosis and/or indirectly cell cycle arrest. (ii) The molecular mechanism that explains p16Ink4a overexpression in HPV-related neoplasms is the presence of viral oncoproteins E6 and E7. Rb protein is inactivated by interaction with the high-risk HPV oncoprotein E7, and oncoprotein E6 induces degradation of the tumor suppressor p53. (iii) Cytoplasmic overexpression of p16Ink4a has been associated with its sequestration by other proteins such as CDK4 or AE1. (iv) The p16Ink4a protein interaction with other proteins like γ2 chain of laminin 5, β-catenin or VEGF seems to be related to new functions attributable to p16Ink4a (inhibition of angiogenesis and cell invasion).

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p16Ink4a and senescence

Cellular senescence is a growth arrest mechanism that protects the cell from hyperproliferative signals and from various forms of stress (Hayflick, 1965; Bringold and Serrano, 2000; Collado et al., 2005). It can be activated during ageing (replicative senescence) or in response to various stress stimuli, such as DNA damage, oxidative stress or exposure to drugs (premature senescence) (Passegue and Wagner, 2000; Krishnamurthy et al., 2004; Chen et al., 2005; Yogev et al., 2006; Ruas et al., 2007). Senescent cells exhibit several changes, including a flattened and enlarged appearance, expression of senescence-associated β-galactosidase activity and senescence-associated heterochromatic foci (Gil and Peters, 2006). Senescence and cell cycle arrest in non-senescent cells seem to share, at least in part, the same molecular mechanisms, involving the p16Ink4a/Rb and p14ARF/p53 pathways (Schmitt et al., 2002).

Expression of p16Ink4a markedly increases with ageing in most mouse tissues and in human skin and kidney tissues (Zindy et al., 1997; Ressler et al., 2006), suggesting the importance of this tumor suppressor in ageing and senescence (Hara et al., 1996; Zhu et al., 2002). In addition, p16Ink4a overexpression has been reported in senescent fibroblasts (Wu et al., 2007), in response to oxidative stress (Ksiazek et al., 2006; Quereda et al., 2007), DNA damage and changes in chromatin structure (Canepa et al., 2007; Fordyce et al., 2010). Nonetheless, a complete understanding of the signals that trigger senescence is currently lacking, and although p16Ink4a appears to be one of the principal factors in senescence, more information is needed to ascertain the exact role of each factor in this process.

Other functions attributed to p16Ink4a

In addition to the action of p16Ink4a in cell cycle regulation, this protein has also been implicated in other processes, such as apoptosis, cell invasion and angiogenesis, and these activities may be related to its overexpression in cancer.

p16Ink4a overexpression has been observed at the invasive front of endometrial, colorectal and basal cell carcinoma (Jung et al., 2001; Natarajan et al., 2003; Svensson et al., 2003; Horree et al., 2007). Supporting the hypothesis of a relationship between p16Ink4a and invasion, overexpression of p16Ink4a in these tumor areas has been associated with other molecules that are shown to be related with invasive status (for example, the γ2 chain of laminin 5 and β-catenin) (Palmqvist et al., 2000; Jung et al., 2001; Natarajan et al., 2003). Furthermore, in vitro studies have shown that p16Ink4a is implicated in the regulation of matrix-dependent cell migration (Fahraeus and Lane, 1999), in glioma invasion (Chintala et al., 1997) and in the inhibition of breast cancer cell migration (Li and Lu, 2010).

Angiogenesis and apoptosis are also associated with p16Ink4a function, but there is little published information on these topics. In brief, p16Ink4a restoration has resulted in vascular endothelial growth factor (VEGF) downregulation in various cell lines and inhibition of angiogenesis in malignant gliomas (Harada et al., 1999). Several reports have provided experimental evidence, indicating that p16Ink4a activity is related to avb3 in melanoma (Fahraeus and Lane, 1999), glioma (Adachi et al., 2001) and pancreatic cancer (Marchan et al., 2010), linking p16Ink4a with this well-known angiogenic integrin (Figure 1).

In addition, induction of apoptosis in a p53-dependent (Kataoka et al., 2000; Katsuda et al., 2002) or independent manner (Calbo et al., 2001, 2004; Modesitt et al., 2001) in response to overexpression of p16Ink4a has been observed in various cancer cell lines. Lastly, p16Ink4a appears to contribute substantially to hematopoiesis, promoting differentiation and apoptosis of erythroid cells by modulating bcl-x and NF-κB functions (Minami et al., 2003).

Of further interest, p16Ink4a has been described in the cytoplasm of some tumor cells where functions other than control of proliferation could take place (Haller et al., 2010), as has also been recently reported by Chien et al. (2010). Considering these data, a role of p16Ink4a as a universal cancer suppressor, acting to block several pro-neoplastic cell capabilities (proliferation, invasion and angiogenesis), can be hypothesized. The p16Ink4a gene seems to be much more than simply a well-recognized regulator of cell cycle progression; hence, further studies are required to expand our knowledge of its possible mission in cancer development and progression.

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p16Ink4a as a tumor suppressor

p16Ink4a is a negative regulator of cell proliferation, and therefore one of the main factors to avert tumor formation. Close to 50% of all human cancers show p16Ink4a inactivation ranging from 25 to 70% (Gonzalez and Serrano, 2006); these include head and neck, esophagus, biliary tract, liver, lung, bladder, colon and breast carcinomas; leukemia; lymphomas; and glioblastomas (Ueki et al., 1996; Serrano, 1997; Chim et al., 2003; Di Vinci et al., 2005). Pancreatic carcinomas constitute the paradigm of tumors presenting p16Ink4a inactivation, with 98% of cases showing a loss of p16Ink4a function (Moore et al., 2001a, 2001b; Fukushima et al., 2002; Bardeesy et al., 2006). In these tumors, several mechanisms whereby the p16Ink4a gene is inactivated have been described, including homozygotic deletions, loss of heterozygosity, point mutations and promoter methylation (Herman et al., 1995; Rutter et al., 2003; Paulson et al., 2008; Andujar et al., 2010).

p16Ink4a inactivation has been reported to be an early and critical event in tumor progression in some types of tumors (Chao et al., 2008; Paulson et al., 2008; Guida et al., 2009; Carnero and Lleonart, 2010). In this sense, certain molecular mechanisms of p16Ink4a repression have been directly associated with carcinogens, such as tobacco in lung cancer (Wang et al., 2005) and oxidative stress due to reactive oxygen species (Tanaka et al., 1999; Hiroyasu et al., 2002), suggesting a relevant role in the development of some preneoplastic lesions. However, p16Ink4a inactivation has also been described as an intermediate or late event in tumor progression in pancreatic carcinoma, the tumor showing the most constant p16Ink4a repression (Fukushima et al., 2002).

Other investigations have shown INK4/ARF locus germline mutations in some familial syndromes, reinforcing the tumor suppressor role of p16Ink4a. For example, missense mutations or deletions of the INK4/ARF locus represent the most important alterations in familial melanoma (41% of cases) (Orlow et al., 2007; Kannengiesser et al., 2009). Furthermore, in families with BRCA2, MMR genes and INK4/ARF locus mutations, there is an increased risk of cutaneous melanoma, acute lymphatic leukemia and blast leukemia in childhood (Magnusson et al., 2008; Daniotti et al., 2009). Similarly, mutations in the INK4/ARF locus gene have been detected in familial atypical mole multiple melanoma syndrome, and in individuals with combined occurrence of pancreatic cancer and melanoma (Bartsch et al., 2002; Ghiorzo et al., 2004; Hruban et al., 2007). Finally, INK4/ARF locus/CDK4 germline mutations associated with BRCA1, BRCA2 and p53 alterations have been described in familial breast carcinoma (Monnerat et al., 2007). All these findings highlight the role of p16Ink4a as a tumor suppressor, but cannot explain p16Ink4a overexpression in tumors, the main objective of this review.

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p16Ink4a overexpression in tumors

A wealth of information has been generated about p16Ink4a as a tumor suppressor protein that is downregulated in cancer. However, p16Ink4a can also be overexpressed in tumors, and few studies have been performed to elucidate this fact, except in the case of human papilloma virus (HPV)-related neoplasms (Milde-Langosch et al., 2001; Garcia et al., 2004; Arifin et al., 2006; Ivanova et al., 2007; O’Neill et al., 2007; Lam et al., 2008). In fact, p16Ink4a overexpression in some types of tumors, such as cervical cancer, head and neck cancer and perianal lesions is used as a diagnostic tool and has been directly associated with infection by high-risk genotypes of HPV (Mulvany et al., 2008). Considering that knowledge of the relationship between p16Ink4a and HPV has been discussed at length within the pathology community, this review will only briefly summarize some important points on this subject, and focus on p16Ink4a overexpression in neoplasms unrelated to HPV.

p16Ink4a overexpression in HPV-related tumors

During immortalization of cancer cells, the p16Ink4a–Rb pathway is often targeted by viral oncoproteins because of its critical activity to prevent inappropriate cell proliferation. One of these oncoproteins is latent membrane protein 1 of Epstein–Barr virus, which blocks p16Ink4a expression (Ohtani et al., 2003). However, most viruses that interact with the p16Ink4a–Rb pathway, like HPV, are associated with p16Ink4a overexpression because of direct or indirect inactivation of Rb (Guenova et al., 1999; Martin et al., 2000). The presence of HPV oncoproteins E6 and E7 is the molecular mechanism that explains p16Ink4a overexpression in these cases. Rb protein is inactivated by interaction with the high-risk HPV oncoprotein E7 (Munger et al., 1989; Huang et al., 1993), and oncoprotein E6 induces degradation of the tumor suppressor p53 (Figure 1). Rb inactivation releases p16Ink4a from its negative feedback control, causing a paradoxical increase in the levels of this protein, which attempts to inhibit uncontrolled cellular replication. In summary, p16Ink4a is overexpressed in HPV-related tumors in an unsuccessful attempt to stop cell proliferation (Reuschenbach et al., 2008).

HPV-related cancers presenting p16Ink4a overexpression are very sensitive to radiotherapy, and have a better prognosis than those unrelated to HPV. In this context, p16Ink4a overexpression has been suggested to have a major impact on treatment response and survival in patients with head and neck cancer treated with conventional radiotherapy (Gupta et al., 2009; Lassen et al., 2009; Fischer et al., 2010), leading to the hypothesis that malignant tumors overexpressing p16Ink4a have higher radiosensitivity.

Subcellular location of p16Ink4a overexpression

Classically, the only function attributed to p16Ink4a has been cell cycle regulation and this function takes place in the nucleus. Surprisingly, there is considerable evidence that several neoplasms exhibit significant p16Ink4a levels in cytoplasm (Evangelou et al., 2004). Moreover, cytoplasmic p16Ink4a has been associated with tumor progression and prognosis in some kinds of neoplasms. For example, in breast cancer, the presence of p16Ink4a was preferentially confined to the nucleus in fibroadenoma and was nuclear/cytoplasmic or exclusively cytoplasmic in carcinoma (Milde-Langosch et al., 2001; Di Vinci et al., 2005). In colorectal cancer, p16Ink4a exhibits a similar pattern, showing strong nuclear/cytoplasmic positivity (about 80%) in adenomas and in primary or metastatic adenocarcinomas, whereas negativity or low nuclear expression is observed in normal mucosa and in benign conditions (Dai et al., 2000; Di Vinci et al., 2005; Zhao et al., 2006). In addition, in non-epithelial tumors such as astrocytomas or uterine leiomyosarcomas, p16Ink4a overexpression can be detected in both the nucleus and the cytoplasm, and this compartmentalized staining has been associated with high-grade malignant phenotypes (Beasley et al., 2003; Arifin et al., 2006; O’Neill et al., 2007). Further supporting this hypothesis, Haller et al. (2010) have recently described the association between p16Ink4a cytoplasmic overexpression and p16Ink4a nuclear downregulation, with poor prognosis in gastrointestinal stromal tumor. Interestingly, nuclear p16Ink4a downregulation was associated with an overexpression of E2F. In contrast, cytoplasmic p16Ink4a overexpression did not exhibit any relation with E2F expression. These results support the hypothesis that p16Ink4a has different roles in different subcellular locations, and that the control of cell cycle is mainly regulated by nuclear p16Ink4a. Notably, other cell cycle regulators usually located in the nucleus have been found to be translocated to cytoplasm in several tumors, such as p27 or PTEN, both proteins having the ability to perform different actions from different subcellular locations (Sanchez-Beato et al., 1999; Lobo et al., 2008; Kim et al., 2009).

Evaluation of the cytoplasmic location of p16Ink4a is a relatively recent event. Currently, p16Ink4a cytoplasmic expression has been considered as background in many studies, whose results should be interpreted with caution (Marsh and Varley, 1998; Milde-Langosch et al., 2001). Several hypotheses to explain the presence of p16Ink4a in cytoplasm have been described, and it seems that cytoplasmic accumulation is not related to an alteration of the p16Ink4a gene (Emig et al., 1998). p16Ink4a phosphorylation induces formation of the p16Ink4a/CDK4 complex (Figure 1), preventing cyclin D binding and subsequent kinase function. This complex is a large molecule that cannot pass through the nuclear membrane (Gump et al., 2003; Zhao et al., 2006). Cytoplasmic localization of p16Ink4a due to CDK4 sequestration has been suggested to occur in neoplastic cells as an indirect phenomenon of an alteration in the Rb pathway (Zhao et al., 2006). Another protein related to aberrant accumulation of p16Ink4a in the cytoplasm is anion exchanger 1 (AE1). AE1 is a transmembrane protein that interacts with p16Ink4a in gastric and colon cancer cells, sequestrating p16Ink4a in the cytoplasm with co-accumulation of both the proteins. Small interfering RNA-mediated silencing of AE1 induces the release of p16Ink4a from the cytoplasm to the nucleus, leading to cell death and inhibition of tumor cell growth (Shen et al., 2007).

In addition, proteomic and post-translational studies have been performed in an attempt to clarify the function of p16Ink4a in the cytoplasm. These studies have shown that p16Ink4a is expressed in the cytoplasm and the nucleus, depending on post-translational modifications or its capability to form a complex with other proteins (Nilsson and Landberg, 2006). p16Ink4a interacts with several proteins located in the cytoplasm, such as α-β-γ actin, α-β tubulin and CDK4/6 (Souza-Rodrigues et al., 2007). Some actions of cytoplasmic p16Ink4a have been suggested, such as dissociation of the αvβ3 integrin from focal adhesions (Fahraeus and Lane, 1999). Nevertheless, further work is needed to elucidate the molecular mechanisms involving cytoplasmic location of p16Ink4a, its functions and its connection with oncogene-induced senescence (OIS) and failure of the p16Ink4a tumor suppressor function.

In conclusion, as occurs with other proteins involved in cell cycle regulation, p16Ink4a localization in the cytoplasm may represent an alternative mechanism for modulating different pathways, instead of simply a way to inactivate its cell cycle control function.

p16Ink4a overexpression in progression of non-HPV-related cancers

The steps that take pre-malignant lesions to malignant transformation include many genetic alterations, mainly involved in activation of proto-oncogenes and inactivation of tumor suppressor genes.

Although p16Ink4a loss is recognized as a critical event in tumor initial progression (Chen et al., 2005; Paulson et al., 2008; Guida et al., 2009; Carnero and Lleonart, 2010), it has also been observed that increased expression of this protein is important for later malignant transformation. Two different patterns of p16Ink4a expression in the progression from normal tissue to malignant tumors have been described: (i) p16Ink4a is overexpressed in benign and pre-malignant lesions, but not in malignant ones and (ii) p16Ink4a is overexpressed in malignant tumors.

p16Ink4a overexpression in benign and pre-malignant lesions: the role of OIS
 

Several sources of evidence have suggested that the ability to bypass senescence is the main molecular mechanism involved in the progression of pre-malignant to malignant cells (Dai et al., 2000; Braig et al., 2005; Collado et al., 2005; Zhang et al., 2006). This hypothesis is based on the concept of oncogene-induced senescence, which was established after demonstration of p53- and p16Ink4a-mediated senescent-like arrest in response to expression of oncogenic Ras in normal primary cells (Collado et al., 2007). This event has been considered as a possible mechanism to prevent proliferation of potentially dangerous cells (Serrano, 1997). Supporting this concept, senescent cells have been shown in a number of different benign lesions, including nevi and neurofibromas (Michaloglou et al., 2005; Courtois-Cox et al., 2006), but not in malignant lesions. p16Ink4a overexpression has been found in premature senescence, and particularly in OIS, and consequently in benign and premalignant lesions with senescent cells (Krishnamurthy et al., 2004; Michaloglou et al., 2005; Gray-Schopfer et al., 2006).

Human nevi are benign proliferations of melanocytes, and represent the paradigm of OIS in human tumors. These lesions remain in a growth-arrested state and only rarely progress to melanoma. A mutation in a downstream effector of Ras, BRAF (BRAFv600E), is often found in these tumors. BRAFv600E expression in human melanocytes induces cell cycle arrest, accompanied by p16Ink4a overexpression and senescence-associated acidic β-galactosidase activity (Michaloglou et al., 2005; Gray-Schopfer et al., 2006). Recent evidence has described a similar situation in schwannomas and neurofibromas (Romagosa et al., 2009). These tumors overexpress p16Ink4a, and show senescence-associated acidic β-galactosidase activity, BRAF mutations and growth arrest, with null or very low proliferative activity (Romagosa et al., 2009; Serrano et al., 2010). Moreover, the malignant counterparts of both tumors, melanoma and malignant peripheral nerve sheath tumor, are immunohistochemically negative for p16Ink4a (Figure 2) (Sabah et al., 2006), and loss of p16Ink4a has been involved in the development of both types of neoplasms (Kourea et al., 1999; Nielsen et al., 1999; Perrone et al., 2003; Sabah et al., 2006). Therefore, some benign tumors overexpress p16Ink4a and this overexpression seems to control proliferation in response to oncogenic stimuli, protecting the cell from malignant transformation. This phenomenon could have a diagnostic application, as in the differential diagnosis from pre-malignant to malignant lesions in malignant peripheral nerve sheath tumor (unpublished data), melanoma (Hilliard et al., 2009) and prostate cancer, where p16Ink4a immunopositivity has been described as a possible additional tool to differentiate between prostate intraepithelial neoplasia and carcinoma (Zhang et al., 2006). However, recent evidence suggests that not all kinds of cells have the ability to enter in senescence when faced with the same stimuli. In fact, recent studies have shown that expression of oncogenic K-RAS in mice induces senescence in colonic cells, while small bowel cells become only hyperplasic (Bennecke et al., 2010; Carragher et al., 2010). These results suggest that something more than oncogenic stimuli is needed to induce senescence, and this secondary event seems to be tissue/cellular background specific.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic representation of molecular basis and uses of p16Ink4a overexpression in human tumors. p16Ink4a overexpression in benign lesions is associated with senescence induction in response to oncogenic stimuli. This situation is represented by (a), which corresponds to a case of Schwannoma with high p16Ink4a expression and very low Ki67 index. Malignant transformation could be associated with a loss of p16Ink4a and its protective role, such as in (b), which corresponds to a case of malignant peripheral nerve sheath tumor showing a negative p16Ink4a immunostaining and a high Ki67 index. Furthermore, p16Ink4a is overexpressed in malignant tumors with Rb alterations and this pattern seems to be associated with high-grade tumors, at least in some tissues. (c) is an example of the last pattern and corresponds to a case of high-grade undifferentiated sarcoma with high p16Ink4a overexpression and very high Ki67 index. All images are at same magnification ( × 20). H&E: Hematoxylin/eosin staining.

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This pattern of p16Ink4a overexpression suggests that p16Ink4a inhibition is the main step in bypassing senescence during malignant transformation. However, in vitro studies have shown that there are other proteins involved in OIS, such as RSK4. Surprisingly, in vitro RSK4-induced senescence is independent of p16Ink4a and p53, but requires p21cip/waf1 and Rb (Lopez-Vicente et al., 2009). More studies are needed to identify other molecules implicated in senescence activation and their exact role in different human tumors.

p16Ink4a overexpression in malignant tumors: alterations of the p16Ink4a–Rb pathway
 

A progressive increase of p16Ink4a expression has been described in the transformation from normal tissue to preneoplastic lesions, and from preneoplastic lesions to carcinoma in several types of cancer (Dai et al., 2000; Milde-Langosch et al., 2001; Di Vinci et al., 2005; Zhao et al., 2006; Hilliard et al., 2009).

In the colon, two different patterns of p16Ink4a overexpression have been observed. The first pattern is related to senescence in serrated adenomas, which have malignant transformation associated with p16Ink4a downregulation (Carragher et al., 2010). The second pattern is characterized by a very low p16Ink4a immunostaining in normal mucosa, with a progressively higher expression in aberrant crypt foci, non-serrated adenomas, primary carcinomas and metastatic tumors (Dai et al., 2000; Zhao et al., 2006). A similar pattern has been observed in skin where p16Ink4a expression increases from relatively low levels in pre-malignant lesions (actinic keratosis) to high levels in in situ and infiltrating carcinomas (Nilsson et al., 2004), and in breast tissues where negative or low expression is seen in normal ductal epithelium, together with a progressive increase in benign lesions and carcinoma (Milde-Langosch et al., 2001; Di Vinci et al., 2005). Furthermore, increased nuclear p16Ink4a protein expression compared with normal epithelium has been demonstrated in preneoplastic and tumor tissues of the gallbladder (Lynch et al., 2008). On the basis of similar sources of evidence, it has been suggested that p16Ink4a can be used in the differential diagnosis of uterine leiomyosarcoma from leiomyoma and smooth muscle tumors of uncertain malignant potential based on p16Ink4a overexpression in leiomyosarcoma (Atkins et al., 2008).

As has been suggested, OIS could have a role in the development of pre-malignant lesions, explaining p16Ink4a overexpression in benign and pre-malignant lesions (Zhang et al., 2006; Campo-Trapero et al., 2008). However, p16Ink4a should stop proliferation in cells with a properly functioning p16Ink4a–Rb pathway. Therefore, alterations in this pathway should be present in cases where a progressive increment of p16Ink4a is seen from pre-malignant lesions to malignant ones.

In this sense, deregulation of Rb results in increased p16Ink4a expression in tumor cells and cancer tissue due to positive feedback (Schwartz et al., 1998). Rb loss is a frequent event in many neoplasms and it is associated with uncontrolled cell proliferation. In a recent report, loss of heterozygosity of Rb was found in 39% of breast tumors (Herschkowitz et al., 2008). In the same samples, high p16Ink4a protein expression was observed, and there was a statistical correlation between Rb loss of heterozygosity and p16Ink4a expression levels (P=0.01). Actually, an inverse relationship between p16Ink4a and Rb expression has been reported in breast cancer (Dublin et al., 1998; Gorgoulis et al., 1998), and recently in lung cancer (Bastide et al., 2009). Moreover, studies by our group have provided evidence of p16Ink4a overexpression in a subgroup of undifferentiated high-grade pleomorphic sarcomas (Figure 2), and Rb loss of heterozygosity was found in 5/6 of these cases (unpublished data). Thus, the relationship between p16Ink4a and Rb could explain overexpression of the p16Ink4a tumor suppressor protein in malignant tumors showing uncontrolled proliferation. The possible prognostic role of the various alterations in this pathway remains unknown.

Less is known about the prognostic value of p16Ink4a in pre-malignant lesions. Kerlikowske et al. (2010) have recently described an association between p16Ink4a overexpression in breast ductal carcinoma in situ and the risk of subsequent DCIS or invasive cancer. In brief, p16Ink4a overexpression together with a high ki67 and COX-2 overexpression was associated with progression to an invasive carcinoma, whereas p16Ink4a overexpression with high ki67 but without COX-2 overexpression was associated with subsequent DCIS. So, in this study, high-risk pre-malignant lesions show p16Ink4a overexpression associated with a high ki67 index, indicating that these cells are high-grade pre-malignant cells, but not senescent cells. These results highlight the idea that something more than p16Ink4a overexpression is needed in malignant transformation, and that the evaluation of ki67 is important to elucidate the role of p16Ink4a overexpression in malignant and pre-malignant lesions.

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p16Ink4a as a prognostic marker

p16Ink4a could be used in differentiating the diagnosis from benign to malignant lesions in tumors showing a progressive increase of its expression. However, most malignant tumors overexpressing p16Ink4a do not display constant overexpression. These differences in p16Ink4a levels in malignant disease seem to indicate bypass of senescence, mainly caused by p16Ink4a alterations (showing negative immunohistochemistry staining for p16Ink4a), or changes in Rb or other proteins within the pathway (showing p16Ink4a overexpression due to positive feedback). Several sources of evidence have suggested that p16Ink4a overexpression could have a prognostic role in the group of tumors with irregular p16Ink4a overexpression (Milde-Langosch et al., 2001; Arifin et al., 2006; Lam et al., 2008). For example, in colon adenocarcinomas, p16Ink4a overexpression correlates with clinical features of a poorer prognosis, such as sex, distal location, tumor grade and stage (Lam et al., 2008). In breast cancer, p16Ink4a overexpression was detected in about 20% of tumors and was significantly associated with unfavorable prognostic indicators, such as high grade and negative estrogen receptor status (Milde-Langosch et al., 2001). In the same study, concurrent overexpression of p73 and p16Ink4a was significantly correlated with the lymph node metastasis, positive immunohistochemistry for p53, vascular invasion and negative progesterone receptors. Also, in some non-epithelial lesions, such as high-grade astrocytoma or gastrointestinal stromal tumors, overexpression of p16Ink4a correlates with a poor prognosis and seems to be an unfavorable prognostic indicator (Arifin et al., 2006; Steigen et al., 2008).

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p16Ink4a and treatment

If OIS serves as a critical barrier for malignant transformation, direct restoration due to overcorrection or ablation of senescence-compromising genes could result in induction of senescence and control of proliferation (Schmitt et al., 2007). Recent reports have shown that cancer can be eliminated through senescence induction by re-activating p53, at least in murine models (Schmitt et al., 2007; Serrano, 2007; Lleonart et al., 2009). In the same way, new therapies to restore p16Ink4a could lead to senescence induction, and therefore elimination of cancer. For example, promoter hypermethylation of p16Ink4a or cytoplasmic p16Ink4a sequestration by AE1 has been identified as changes that allow malignant transformation. Therefore, demethylating therapies or inhibition of p16Ink4a sequestering proteins (AE1) could restore p16Ink4a and induce premature senescence in cancer cells (Schmitt et al., 2007; Schwabe and Lubbert, 2007; Shen et al., 2007; Crea et al., 2009).

It has also been shown that DNA-damaging and differentiation-inducing anticancer therapies can provoke senescence as part of their anticancer effect (Schmitt et al., 2007). This means that p16Ink4a overexpression together with a low proliferative index in cancer cells previously treated with this kind of therapy may only be showing senescence induced by the treatment. Consequently, future studies are needed to develop new therapies based on senescence induction, and p16Ink4a is one of the main candidates to work with. A deep knowledge of p16Ink4a–Rb pathway status will be necessary to determine the sensitivity to these therapeutic approaches. In addition, p16Ink4a together with other senescence features could be used as biomarkers to evaluate therapeutic response, assessing arrest in G0 instead of tumor cell destruction, and thus predicting the patient's prognosis.

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Conclusion

p16Ink4a overexpression in human tumors can be indicative of two main situations (Figure 2): p16Ink4a overexpression in benign or pre-malignant lesions, in which the overexpression is secondary to OIS; and p16Ink4a overexpression in malignant lesions, in which overexpression appears to be a mechanism to arrest the uncontrolled proliferation caused by failure of the Rb pathway (secondary to viral infection, mutational silencing of the Rb gene, or other mechanisms). In this review, we have shown evidence of the role of p16Ink4a in cancer diagnosis and prognosis in specific tumors. This evidence allows us to hypothesize that p16Ink4a could be a diagnostic tool, differentiating benign lesions from malignant ones in all tumors in which malignant transformation is associated with p16Ink4a loss, and it could also be a prognostic tool in those tumors in which malignant transformation is sometimes associated with p16Ink4a overexpression due to pRb failure. Better understanding of p16Ink4a overexpression in human tumors will facilitate the search for new uses of p16Ink4a immunohistochemistry as a diagnostic or prognostic tool. Apart from this, there is very little available information regarding the subcellular location of p16Ink4a, and this factor could have important implications on the evaluation of p16Ink4a overexpression in different kinds of tumors. At last, because p16Ink4a is one of the most important tumor suppressor proteins (together with p53), new anticancer therapies involving restoration of p16Ink4a functionality are being investigated. In this context, accurate knowledge of p16Ink4a expression in human tumors is essential for guiding researchers to find new solutions for cancer patients.

In summary, p16Ink4a is currently one of the most promising tumor suppressor genes, and awareness of its level of expression and subcellular location in all different kinds of cancers could be relevant to establishing histological diagnosis of tumors, to predict prognosis and to guide new therapies research.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We are grateful to M Serrano for his careful reading and suggestions, and C Cavallo and S Illing for English editing. SRC is supported by grants from the ‘Fondo de Investigaciones Sanitarias’ (Ref.05/0818 and 08/0143), ‘Fundació Marató TV3’ (Ref.052710), ‘Mutua Madrileña’ (FMMA/2009/02), ‘Redes temáticas de Investigación Cooperativa en Salud’ (Ref.RD06/0020/0104 and RD06/0020/1020) and ‘Generalitat de Catalunya’ (Ref.2005SGR00144), AM is supported by grants from the ‘Ministerio de Ciencia y Tecnología (Ref. BIO2008-04692-C03-03) and Generalitat de Catalunya (Ref 2009SGR624), and CR is supported by a grant from the ‘Grupo español de Investigación en Sarcomas’ (Ref.GEIS/2008).