p16, The Cyclin-Dependent Kinase Inhibitor in Prostate Cancer
By Cheryl T. Lee, and Carlos Cordon-Cardo
The ink4a gene maps to the 9p21 region, and was initially described as encoding a 148 amino acid protein termed p16. The p16 protein exclusively associates with Cdk4 and Cdk6, inhibiting their complexation with D-type cyclins, and the consequent phosphorylation of the product encoded by the retinoblastoma gene (RB), pRB. This interaction contributes to cell cycle arrest. In prostate cancer, the role of p16 has not been well elucidated, though analyses utilizing microsatellite markers in the vicinity of the INK4A gene have revealed loss of heterozygosity in a subset of primary and metastatic prostate tumors. However, unlike other primary tumors, p16 inactivation, either through deletions, mutations, or through promoter methylation, appears to be an infrequent event in prostate cancer. Nevertheless, we have recently observed that a subset of prostate cancer patients demonstrates overexpression of p16. Moreover, these patients have a poorer clinical course. The mechanism for this association has not been fully elucidated, although androgen depletion and/or alterations in the RB pathway may contribute to the inactivation of p16-mediated tumor suppressor activities.
p16
The INK4A gene maps to the short arm of chromosome 9 (9p21) and was initially described as encoding a protein of Mr 15,845, termed p16.1,2 The p16 protein forms binary complexes exclusively with Cdk4 and Cdk6, inhibiting their kinase activity and subsequent pRB phosphorylation during the G1 phase of the cell cycle.1,3 Additional complexity results from the presence of a second INK4A product termed p19ARF.4 The p19ARF protein has recently been shown to interact with mdm2 and to block mdm2-induced p53 degradation and transactivational silencing.5 The two products, p16 and p19ARF, share exons 2 and 3 of the INK4A gene, but have distinct promoters and exon 1 units: exon 1a (p16) and exon 1b (p19ARF). The INK4A-a gene encodes p16 and is mutated in a wide variety of tumor cell lines and certain primary tumors.2 In addition, methylation of the 5’ CpG island of the exon 1a promoter region is a frequent mechanism of p16 inactivation in primary tumors.6
p16 and Prostate Cancer
The precise role of p16 in prostate cancer development and progression is not well understood. Unlike other primary tumors and cell lines, p16 inactivation, either through deletions, mutations, or promoter methylation, appears to be an infrequent event in prostate cancer.7-12 Homozygous deletions of the INK4A-a gene do not occur in six of the prostate cancer cell lines available, including LNCaP, PC3, DU145, TSU-Pr1, PPC-1, and DuPro-18,9,11,13 However, a missense mutation has been reported for DU145 cells.9,11 As stated above, it is known that the transcription of the INK4A-a gene can be inhibited by promoter methylation. In PC3, TSU-Pr1, and DuPro-1 cells, lack of p16 mRNA expression has been associated with methylation of the promoter region of the INK4A exon 1a, effectively inactivating p16.11,13 Induction of p16 mRNA was subsequently accomplished by treating these cells with 5-Aza-2’-deoxycytidine, a demethylating agent acting through inhibition of 5-methyltransferase. In contrast, LNCaP cells were reported to have an unmethylated INK4A exon 1a and expressed a p16 mRNA product.11,13,14
In primary prostate tumors, mutations and deletions of the INK4A-a gene are also infrequent, with alterations reported in 0-6% of cases.7-10,12 However, microsatellite analyses utilizing markers in the vicinity of the INK4A gene, have revealed loss of heterozygosity in a subset of 12 of 60 (20%) primary and 13 of 28 (46%) metastatic prostate tumors.11 The significance of this finding is not clear, as the 9p21 locus is quite complex, producing at least three tumor suppressor genes: 1) the INK4A-a product, p16; 2) the INK4A-b product, p19ARF; and 3) the INK4B product, p15, another cyclin-dependent kinase inhibitor. Unlike prostate cancer cell lines, promoter methylation appears to be an uncommon event; DNA extracted from non-microdissected primary tumors revealed methylation in only 5-12% of cases.11,12
p16 Expression
We have recently reported the patterns of p16 expression in normal and malignant tissues, including 88 primary tumors. This study was conducted using in situ hybridization and immunohistochemistry assays in order to determine the status of the INK4A exon 1a transcripts and levels of p16 protein, respectively.15 Associations between altered p16 phenotypes and clinicopathologic variables were also studied to further define their potential implications in prostate cancer. Clinicopathologic variables included pre-treatment prostate-specific antigen (PSA) level, Gleason grade, pathologic stage, hormonal status, and biochemical (PSA) relapse after surgery.
We found that the levels of p16 expression and INK4A exon 1a transcripts in normal prostate and benign hyperplastic tissues were undetectable. However, two distinct p16 phenotypes were observed in primary prostatic adenocarcinomas. Most tumors were found to have undetectable or very low levels of p16 protein expression (Group A, 57% of cases). This was associated with low levels or absence of INK4A exon 1a transcripts. Another group of tumors showed elevated p16 protein expression (Group B, 43%), which was consistently associated with increased INK4A exon 1a transcripts. Based on these results, we concluded that upregulation of the INK4A-a gene led to p16 protein overexpression. Overexpression of p16 was associated with a higher pre-treatment PSA level (P = 0.018), the use of neoadjuvant androgen ablation (P = 0.001), and a sooner time-to-PSA relapse after radical prostatectomy (P = 0.002). A trending association of p16 overexpression with higher pathologic stage was also observed (P = 0.087). These data suggest that p16 overexpression is associated with tumor recurrence and a poor clinical course in prostate cancer patients. In support of this postulate, another study dealing with prostate cancer has also reported an association between p16 overexpression and poor outcome, as related to biochemical failure.16
The expression data demonstrate that normal prostate tissue and most primary tumors have undetectable levels of p16, both at the protein and transcript levels.15 This negative phenotype could not be explained by molecular analyses, since the INK4A-a gene is infrequently altered in prostate cancer. As a matter of fact, it can be deduced that the negative phenotype observed in most primary tumors corresponds to the normal physiologic state of p16. This is further supported by our observation that prostate cancer patients with low-to-undetectable p16 have a less aggressive behavior.15 This is not the case in other neoplastic diseases, since it has been reported that certain tumors with diminished p16 protein levels, such as non-small cell lung cancer, lymphoma and melanoma, tend to have a more aggressive clinical course.17-19
The upregulation of the INK4A-a gene, resulting in the overexpression of p16 protein, may develop through different mechanisms. An association between increased p16 transcript and protein levels occurs in tumor cell lines and certain primary neoplasms that lack functional pRB.1,20 Moreover, p16-mediated inhibition of cell cycle progression appears to be dependent upon functional pRB. These data support an association between p16 and pRB, where absence of functional pRB limits p16 activity and possibly promotes INK4A-a upregulation. Alternatively, enhanced activation of the INK4A-a gene may occur. E2F1, a direct activator of the INK4A exon 1b promoter, does not directly activate INK4A-a transcription.22 However, evidence does exist for an indirect effect, as E2F1 overexpression has been reported to markedly increase p16 transcripts and p16-related cyclin-dependent kinase inhibitor activity. Overexpression of cyclin D1 and/or Cdk4 may also influence p16 expression, through a compensatory feedback loop where deregulation of cyclin D/Cdk4 complexes results in increased levels of p16 protein.24,25 In summary, it appears that an altered RB pathway could trigger p16 overexpression in certain tumors.
Cellular Stress may Trigger Overexpression
Cellular stress produced by altered androgen levels may also trigger p16 overexpression. In prostate cancer patients treated with neoadjuvant androgen ablation prior to radical prostatectomy, overexpression of p16 protein was observed in 71% of hormone-treated vs. 26% of hormone-naïve patients (P = 0.001).15 These data suggest that p16 expression may be enhanced by androgen depletion. Androgens are known to modulate the expression of other cyclin-dependent kinases, such as p27/KIP1 and p21/WAF1.26 In addition, it has been reported that the presence of androgens triggers downregulation of p16 in LNCaP cells—a finding consistent with the observation that p16 overexpression occurs at a greater frequency in cases of androgen ablation.27
The successful transduction of p16 using adenoviral vectors has demonstrated a reduction in the viability of prostate cancer cells in vitro, as well as a decrease in the growth of prostate tumor implants in nude mice. PC3 cells transfected with a p16 expression vector underwent a 70% reduction in cell number when compared with parental and control vector-transfected PC3 cells.28 Similarly, when PPC-1 tumor cells implanted into nude mice were treated with a p16 expression vector, a reduction in tumor size and longer animal survival time were demonstrated.
Clinical Significance
The clinical significance of p16 in prostate cancer is not yet elucidated. Point mutations or deletions are infrequent causes of p16 inactivation. However, p16 overexpression appears to represent an altered phenotype, which identifies a subgroup of patients with a higher likelihood of post-surgical recurrence. Though p16 acts as a negative cell cycle regulator, specific mechanisms may contribute to its altered expression, overcoming p16-mediated tumor suppressor activities. Future studies are necessary to better understand the mechanism of p16 overexpression relative to androgen depletion. Clinical trials aimed at transducing wild-type tumor suppressor genes, such as p53 and p16, are being pursued. It is too early to foresee the impact of such novel therapies in patient management. As in any emerging field, obstacles impose limitations that are usually overcome with experience and advances in technology. (Dr. Lee is Fellow, Urology Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY; Dr. Cordon-Cardo is Director, Division of Molecular Pathology, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY.)
References
1. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell cycle control causing specific inhibition of cyclinD-cdk4. Nature 1993;366:704-707.
2. Kamb A, Gruis N, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436-440.
3. Quelle D, Ashmun R, Hannon G, et al. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene 1995;11:635-645.
4. Quelle D, Zindy F, Ashmun R, et al. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 1995;83:993-1000.
5. Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell 1998;92:713-723.
6. Merlo A, Herman J, Mao L, et al. 5’ CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/Mts1 in human cancers. Nature Med 1995;1:686-692.
7. Mangold K, Takahashi H, Brandigi C, et al. p16 (CDKN2/MTS1) gene deletions are rare in prostatic carcinomas in the United States and Japan. J Urol 1997;157:1117-1120.
8. Park D, Wilczynski S, Pham E, et al. Molecular analysis of the INK4 family of genes in prostate carcinomas. J Urol 1997;157:1995-1999.
9. Tamimi Y, Bringuier P, Smit F, et al. p16 mutations/ deletions are not frequent events in prostate cancer. Brit J Cancer 1996;74:120-122.
10. Chen W, Weghorst M, Sabourin C, et al. Absence of p16/MTS1 gene mutations in human prostate cancer. Carcinogenesis 1996;17:2603-2607.
11. Jarrard D, Bova S, Ewing C, et al. Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer. Genes Chrom Cancer 1997;19:90-96.
12. Gu K, Mes-Masson AM, Gauthier Jet al. Analysis of the p16 tumor suppressor gene in early-stage prostate cancer. Mol Carcinogenesis 1998;21: 164-170, 1998.
13. Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Can Res 1995;55:4525-4530.
14. Chi SG, deVere White RW, et al. Frequent alteration of CDKN2 (p16INK4A/MTS1) expression in human primary prostate carcinomas. Clin Can Res 1997;3:1889-1897.
15. Lee CT, Capodieci P, Osman I, et al. Overexpression of the cyclin-dependent kinase inhibitor p16 is associated with tumor recurrence in human prostate cancer. In Press, Clin Can Res; 1999.
16. Halvorsen OJ, Hostmark J, Haukass, et al. Prognostic importance of p16 and CDK4 proteins in localized prostate cancer. Proc Am Assoc Cancer Res 1997;38:526.
17. Taga S, Osaki T, Ohgami A, et al. Prognostic value of the immunohistochemical detection of p16INK4 expression in nonsmall cell lung carcinoma. Cancer 1997;80:389-395.
18. Garcia-Sanz R, Gonzalez M, Vargas M, et al. Deletions and rearrangements of cyclin-dependent kinase 4 inhibitor gene p16 are associated with poor prognosis in B cell non-Hodgkin’s lymphomas. Leukemia 1997;11:1915-1920.
19. Straume O, Akslen L. Alterations and prognostic significance of p16 and p53 protein expression in subgroups of cutaneous melanoma. Int J Cancer 1997;74:535-539.
20. Parry D, Bates S, Mann DJ, et al. Lack of cyclin D-cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J 1995;14:503-511.
21. Lukas J, Parry D, Aagaard L, et al. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature 1995;375:503-506.
22. Robertson K, Jones P. The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Mol Cell Biol 1998;18:6457-6473.
23. Khleif S, DeGregori J, Yee C, et al. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity. Proc Natl Acad Sci U S A 1996;93:4350-4354.
24. Yao J, Pollock R, Lang A, T et al. Infrequent mutation of the p16/MTS1 gene and overexpression of cyclin-dependent kinase 4 in human primary soft-tissue sarcoma. Clin Cancer Res 1998;4:1065-1070.
25. Burns K, Ueki K, Jhung S, et al. Molecular genetic correlates of p16 cdk4, and pRb immunohistochemistry in glioblastomas. J Neuropathol Exp Neurol 1998;57:122-130.
26. Kokontis J, Hay N, Liao S. Progression of LNCaP prostate tumor cells during androgen deprivation: Hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol Endocrinol 1998;12:941-953.
27. Lu S, Tsai SY, Tsai M-J. Regulation of androgen-dependent prostatic cancer cell growth: Androgen regulation of CDK2, CDK4, and CKI p16 genes. Cancer Res 1997;57:4511-4516.
28. Lu Y, Zhang Y, Faroog F, et al. Adenoviral vector containing wild type p16 suppresses prostate cancer growth and prolongs survival. Proc Am Assoc Can Res 1999;40:482:(abstr 3183).
The protein product of the p16 gene:
a. complexes with Cdk4 and Cdk6.
b. inhibits complex formation of Cdk4 and Cdk6 with D-type cyclines.
c. inhibits phosphorylation of the RB protein.
d. All of the above
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