Oncogene (2011) 30, 2087–2097 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors C Romagosa1, S Simonetti2,LLo´pez-Vicente1, A Mazo3, ME Lleonart1, J Castellvi1 and S Ramon y Cajal1 1Pathology Department, Fundacio´ Institut de Recerca, Hospital Universitari Vall d’Hebron, Universitat Auto`noma de Barcelona, Barcelona, Spain; 2Pangaea Biotech, Oncology Laboratory, USP Dexeus University Institute, Barcelona, Spain and 3Department of Biochemistry and Molecular Biology, University of Barcelona, Institute of Biomedicine (IBUB), Barcelona, Spain p16Ink4a is a protein involved in regulation of the cell cycle. Physiological role of p16Ink4a Currently, p16Ink4a is considered a tumor suppressor protein because of its physiological role and downregulated expres- p16Ink4a and the cell cycle sion in a large number of tumors. Intriguingly, overexpression p16Ink4a is the principal member of the Ink4 family of p16Ink4a has also been described in several tumors. This of CDK inhibitors. It is codified by a gene localized review attempts to elucidate when and why p16Ink4a over- on chromosome 9p21 within the INK4a/ARF locus, expression occurs, and to suggest possible implications of which encodes for two different proteins with different p16Ink4a in the diagnosis, prognosis and treatment of cancer. promoters: p16Ink4a and p19ARF. Both proteins have Oncogene (2011) 30, 2087–2097; doi:10.1038/onc.2010.614; antiproliferative biological activity, and are involved in published online 7 February 2011 the retinoblastoma protein (Rb) and p53 pathways, respec- tively (Serrano, 1997; Weber et al., 2000; Pei and Xiong, Keywords: p16Ink4a; overexpression; cancer; senescence; 2005). These proteins and their interactions are crucial high grade 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 Ink4a Introduction inhibition is shown in Figure 1. Briefly, p16 binds to CDK4/6, inhibiting cyclin D–CDK4/6 complex Mammalian cells have developed complex mechanisms formation and CDK4/6-mediated phosphorylation of Ink4a against mutagens and inappropriate growth stimuli as Rb family members. Expression of p16 maintains the protection from malignant transformation and tumor- Rb family members in a hypophosphorylated state, igenesis. When irreversible tumor-producing stimuli are which promotes binding to E2F1 and leads to G1 cell present, these defense systems enable cells to take roads cycle arrest (Serrano, 1997). However, this classically other than proliferation, such as apoptosis or senescence known function seems to be just a simplified scheme of Ink4a (Lleonart et al., 2009). Several tumor suppressor genes the global role of p16 , and many aspects of its involved in related pathways regulate the outcome of function and regulation are still partially unresolved. cell development. One of these is p16Ink4a (Alcorta et al., 1996), the focus of this review. p16Ink4a and senescence Ink4a p16 expression has been evaluated in several Cellular senescence is a growth arrest mechanism that tumor types with very different results, ranging from protects the cell from hyperproliferative signals and its loss or downregulation (Brambilla et al., 1999a, b; from various forms of stress (Hayflick, 1965; Bringold Schneider-Stock et al., 2005; Ayhan et al., 2010) to its and Serrano, 2000; Collado et al., 2005). It can be clear overexpression (Milde-Langosch et al., 2001; activated during ageing (replicative senescence) or in Armes et al., 2005; Zhao et al., 2006; Angiero et al., response to various stress stimuli, such as DNA damage, 2008; Buajeeb et al., 2009). The main objective of this oxidative stress or exposure to drugs (premature review is to clarify these apparently contrasting expres- senescence) (Passegue and Wagner, 2000; Krishna- sion patterns and to reveal their implications in cancer murthy et al., 2004; Chen et al., 2005; Yogev et al., management. 2006; Ruas et al., 2007). Senescent cells exhibit several changes, including a flattened and enlarged appearance, expression of senescence-associated b-galactosidase ac- Correspondence: Dr S Ramon y Cajal, Pathology Department, tivity and senescence-associated heterochromatic foci Fundacio´Institut de Recerca, Hospital Universitari Vall d’Hebron, (Gil and Peters, 2006). Senescence and cell cycle arrest in Passeig de la Vall d’Hebron 119-129, Barcelona 08035, Spain. E-mail: [email protected] non-senescent cells seem to share, at least in part, the Ink4a Received 11 July 2010; revised 5 December 2010; accepted 7 December same molecular mechanisms, involving the p16 /Rb 2010; published online 7 February 2011 and p14ARF/p53 pathways (Schmitt et al., 2002). p16Ink4a overexpression in cancer C Romagosa et al 2088 Cell invasion Laminin 5 Angiogenesis γ2 chain α v3 integr DNA damage, mitogenic in inhibition β-catenin stimulation, oxidative stress, etc. VEGF P16Ink4a INK4a/ARF Cytoplasmic Ink4a ARF sequestration P16 P14 P16Ink4a AE1 Mdm-2 Cyclin D1 CDK4 E6 CDK4 P16Ink4a P53 E7 P53 P P P Rb Rb cyp1 Apoptosis E2F PP+ Rb P21 E2F Cytoplasm Nucleus G0 M G1 G2 S Figure 1 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 g2 chain of laminin 5, b-catenin or VEGF seems to be related to new functions attributable to p16Ink4a (inhibition of angiogenesis and cell invasion). Expression of p16Ink4a markedly increases with ageing Jung et al., 2001; Natarajan et al., 2003). Furthermore, in most mouse tissues and in human skin and kidney in vitro studies have shown that p16Ink4a is implicated tissues (Zindy et al., 1997; Ressler et al., 2006), in the regulation of matrix-dependent cell migration suggesting the importance of this tumor suppressor in (Fahraeus and Lane, 1999), in glioma invasion (Chintala ageing and senescence (Hara et al., 1996; Zhu et al., et al., 1997) and in the inhibition of breast cancer cell 2002). In addition, p16Ink4a overexpression has been migration (Li and Lu, 2010). reported in senescent fibroblasts (Wu et al., 2007), in Angiogenesis and apoptosis are also associated with response to oxidative stress (Ksiazek et al., 2006; p16Ink4a function, but there is little published information Quereda et al., 2007), DNA damage and changes in on these topics. In brief, p16Ink4a restoration has resulted chromatin structure (Canepa et al., 2007; Fordyce et al., in vascular endothelial growth factor (VEGF) down- 2010). Nonetheless, a complete understanding of the regulation in various cell lines and inhibition of signals that trigger senescence is currently lacking, and angiogenesis in malignant gliomas (Harada et al., although p16Ink4a appears to be one of the principal 1999). Several reports have provided experimental factors in senescence, more information is needed to evidence, indicating that p16Ink4a activity is related to ascertain the exact role of each factor in this process. avb3 in melanoma (Fahraeus and Lane, 1999), glioma (Adachi et al., 2001) and pancreatic cancer (Marchan Other functions attributed to p16Ink4a et al., 2010), linking p16Ink4a with this well-known angio- In addition to the action of p16Ink4a in cell cycle genic integrin (Figure 1). regulation, this protein has also been implicated in In addition, induction of apoptosis in a p53-depen- other processes, such as apoptosis, cell invasion and dent (Kataoka et al., 2000; Katsuda et al., 2002) or angiogenesis, and these activities may be related to its independent manner (Calbo et al., 2001, 2004; Modesitt overexpression in cancer. et al., 2001) in response to overexpression of p16Ink4a has p16Ink4a overexpression has been observed at the been observed in various cancer cell lines. Lastly, p16Ink4a invasive front of endometrial, colorectal and basal cell appears to contribute substantially to hematopoiesis, carcinoma (Jung et al., 2001; Natarajan et al., 2003; promoting differentiation and apoptosis of erythroid Svensson et al., 2003; Horree et al., 2007). Supporting cells by modulating bcl-x and NF-kB functions (Minami the hypothesis of a relationship between p16Ink4a and et al., 2003). invasion, overexpression of p16Ink4a in these tumor areas Of further interest, p16Ink4a has been described in the has been associated with other molecules that are shown cytoplasm of some tumor cells where functions other to be related with invasive status (for example, the g2 than control of proliferation could take place (Haller chain of laminin 5 and b-catenin) (Palmqvist et al., 2000; et al., 2010), as has also been recently reported by Chien Oncogene