BIOLOGY OF NEOPLASIA

Role of the p16 Tumor Suppressor Gene in Cancer By William H. Liggett, Jr and David Sidransky Since its discovery as a CDKI (cyclin-dependent kinase inhibitor) in 1993, the tumor suppressor p16 (INK4A/ MTS-I/CDKN2A) has gained widespread importance in cancer. The frequent mutations and deletions of p16 in human cancer cell lines first suggested an important role for p16 in carcinogenesis. This genetic evidence for a causal role was significantly strengthened by the observation that p16 was frequently inactivated in familial melanoma kindreds. Since then, a high frequency of p16 gene alterations were observed in many primarytumors. In human neoplasms, p16 is silenced in at least three ways: homozygous deletion, methylation of the promoter, and point mutation. The first two mechanisms comprise the majority of inactivation events in most primary tumors. Additionally, the loss of p16 may be an early event in cancer progression, because deletion of at least one copy is quite high in some

THE CELL CYCLE is a complex circuit composed of

positive and negative protein regulators, the role of which is to duplicate DNA precisely during S phase and to segregate it equally into two identical progeny during M phase.' When a cell leaves the quiescent state of Go and enters a metabolically active phase during G 1, the fate of the cell cycle hinges in the balance as the decision to undergo division must be made at the restriction point. Because G1 is such a critical phase of the cell cycle, it is not surprising that many oncogenic perturbations have been found as targeted amplifications or mutations of Gi-specific protein regulators.2, 3 p16 (also known as MTS-1 for major tumorsuppressor 1, INK4a for inhibitor of cyclin-dependent kinase 4a, and CDKN2A or cyclin-dependent kinase inhibitor 2A) is a Gi-specific cell-cycle regulatory gene. The human gene is located on chromosome 9p21, a frequent site of allelic loss (LOH) in many human malignancies. 4-6 The p16 gene is composed of three exons, which encode a 156 amino acid, 15.8 kd protein that blocks progression through the cell cycle by binding to either cyclin-dependent kinase (CDK) 4 or 6 and inhibiting the action of cyclin D. 5,7 The major function of D cyclins is to drive the cell cycle forward by binding to CDKs and forming a catalytically active complex that phosphorylates the pRB protein, which results in release of E2 F and new transcription of important cell regulatory genes. p16 is frequently inactivated in many human cancers, unlike other related INK4 proteins, which also function as CDKIs, and include p15, p18, and p19. Thus, the major biochemical effect of p16 is to halt cell-cycle progression at the GI/S boundary and the loss of p16 function may lead to

premalignant lesions. p 16 is a major target in carcinogenesis, rivaled in frequency only by the p53 tumorsuppressor gene. Its mechanism of action as a CDKI has been elegantly elucidated and involves binding to and inactivating the cyclin D-cyclin-dependent kinase 4 (or 6) complex, and thus renders the retinoblastoma protein inactive. This effect blocks the transcription of important cell-cycle regulatory proteins and results in cell-cycle arrest. Although p16 may be involved in cell senescence, the physiologic role of p16 is still unclear. Future work will focus on studies of the upstream events that lead to p16 expression and its mechanism of regulation, and perhaps lead to better therapeutic strategies that can improve the clinical course of many lethal cancers. J Clin Oncol 16:1197-1206. © 1998 byAmerican Society of Clinical Oncology.

cancer progression by allowing unregulated cellular proliferation. GENETIC EVIDENCE p16 is frequently mutated in patients with familial melanoma and in sporadic cutaneous malignant melanomas. Defects in p16 were first described by two independent groups in many human cell lines.4, 5 These investigators reported a high frequency of homozygous deletion and intragenic mutation of p16 in these cell lines, which provided strong genetic evidence for the role of p16 as a tumor-suppressor gene. The subsequent genomic organization of p16 was elucidated and revealed that p16 was composed of three coding exons: exon 1 (125 bp), exon 2 (307 bp), and exon 3 (12 bp) (Fig 1). A high frequency of p16 deletions were documented in melanoma, esophageal,

From the Department of Otolaryngology-Headand Neck Surgery and The Johns Hopkins Oncology Center, Johns Hopkins Hospital, Baltimore,MD.

Submitted May 12, 1997; acceptedJune 30, 1997. Supported in part by a collaborative agreement with Oncor Inc, Gaithersburg,MD; and grant no. CA-58184-01 from the Lung Cancer Specialized Program of Research Excellence, National Cancer Institute, Atlanta, GA. Address reprint requests to David Sidransky, MD, Johns Hopkins Hospital, Head and Neck Cancer Research Division, 818 Ross Research Building, 720 Rutland Ave, Baltimore, MD 21205-2195; Email [email protected]. © 1998 by American Society of ClinicalOncology. 0732-183X/98/1603-0051$3.00/0

Journalof Clinical Oncology,Vol 16, No 3 (March), 1998: pp 1197-1206

Downloaded from ascopubs.org by 37.44.207.86 on January 27, 2017 from 037.044.207.086 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1197

1198

LIGGETT AND SIDRANSKY

Fig 1. Genomic organization of p 15, p 16p, and p16. p16 is composed of three exons: exon Ia, 2, and 3, which are transcribed to give an mRNA species. p 16P is composed of exons 1g3(upstream of exon la), 2, and 3, and its transcript is an alternative mRNA species.

lung, pancreas, mesothelioma, bladder, head and neck, breast, acute lymphocytic leukemia, brain, osteosarcoma, ovarian, and renal cell lines. 4,5,8 Furthermore, the frequency of inactivation was extraordinarily high and approached 85% in some cell lines. The ubiquitous and frequent inactivation of p16 led to the hypothesis that selective loss of p16 must provide a selective cellular growth advantage to many human tumors. The role of p16 in cancer was further strengthened when investigators studied families with cutaneous malignant melanoma or familial atypical multiple mole/melanoma (FAMM) from the United States, Europe, and Australia. In the familial form of melanoma, which comprises approximately 5% to 10% of cases, approximately half of the identified families showed linkage to markers on chromosomal band 9p21. 9,10 Germline point mutations in p16 were reported in families with linkage to 9p21. In 13 of 18 American FAMM kindreds, eight germline substitutions of p16 were found and at least six of these were likely to be disease-related, and segregated with the disease (ie, 33 of 36 melanomas)." Moreover, 69% (33 of 48) of the individuals in kindreds that carried a pl6 mutation had developed at least one primary melanoma and a majority of the remaining family members had not reached the average age of onset for

the disease. In Dutch FAMM kindreds, 13 of 15 had 19 base pair deletion in p16, apparently a founder mutation, because all 13 families originated from the same geographic location.' 2 Besides melanoma, pancreatic adenocarcinoma is common in some FAMM families and p16 mutations may increase the risk of adenocarcinoma by as much as 13-fold.13 Thus, p16 is frequently mutated in the germline of FAMM families, consistent with the hypothesis that p16 is the familial melanoma gene. In addition to melanoma, p16 point mutations occur commonly in other malignancies, which include pancreatic adenocarcinomas, esophageal carcinomas, and significantly, although less commonly, non-smallcell lung carcinomas and head and neck squamous cell carcinomas (Table 1). MECHANISMS OF INACTIVATION Controversy arose as to whether p16 inactivation was critical only in rapidly dividing cells in tissue culture or in primary human malignancies, because homozygous deletions or intragenic mutations were detected infrequently in primary cancers with LOH at 9p21.6 This controversy was laid to rest by the discovery of microdeletions that encompassed p16 that were difficult to confirm because of the presence of normal cells within primary tumor specimens.

Downloaded from ascopubs.org by 37.44.207.86 on January 27, 2017 from 037.044.207.086 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1199

ROLE OF THE p16 TUMOR SUPPRESSOR GENE IN CANCER Table 1. Frequency of p16 Inactivation in Primary Tumors by Mechanism HD*

Mutation

Methylationt

Tumor Type

%

Range

%

Range

%

Range

Total, %t

Reference

Glioma Head and neck Esophagus Pancreas Bladder Mesothelioma ALL, T-cell Breast NSCLC Colon Melanoma Ovary Prostate Renal cell Pituitary Sarcoma Gastric SCLC

60 50 20 30 45 50 50 10 20 0 25 10 20 15 15 10 10 5

33-85 25-66 0-16 10-37 10-45 21-70 11-70 NR 7-20 0 5-25 3-14 NR NR NR 8-25 5/55 NR