Mechanisms of Inactivation of P14arf, P15ink4b, and P16ink4a Genes in Human Esophageal Squamous Cell Carcinoma1
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2704 Vol. 5, 2704–2713, October 1999 Clinical Cancer Research Advance and Briefs Mechanisms of Inactivation of p14ARF, p15INK4b, and p16INK4a Genes in Human Esophageal Squamous Cell Carcinoma1 Eric Poe Xing,2 Yan Nie,2 Yunlong Song, rations in the p16INK4a locus. Our results show that in hu- ARF Guang-Yu Yang, Yuyang Christine Cai, man ESCCs, p14 is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot Li-Dong Wang, and Chung S. Yang3 of hypermethylation of the 9p21 gene cluster. The frequent Laboratory for Cancer Research, College of Pharmacy, Rutgers, The inactivation of the p14ARF and p16INK4a genes may be an State University of New Jersey, Piscataway, New Jersey 08854-8020 [E. P. X., Y. N., Y. S., G-Y. Y., Y. C. C., C. S. Y.], and Henan important mechanism for the dysfunction of both the Rb Medical University, Zhengzhou, Henan, 457500 China [L-D. W.] and p53 growth regulation pathways during ESCC develop- ment. Abstract Introduction The 9p21 gene cluster, harboring growth suppressive The 9p21 chromosomal band is one of the most frequently genes p14ARF, p15INK4b, and p16INK4a, is one of the major altered genomic regions in human cancers (1). Within a short aberration hotspots in human cancers. It was shown that distance of ;50 kb, this region harbors a gene cluster consisting of p14ARF and p16INK4a play active roles in the p53 and Rb three genes, p14ARF, p15INK4b, and p16INK4a, all of which have tumor suppressive pathways, respectively, and p15INK4b is a putative tumor suppressor roles (2). In addition to physical prox- mediator of the extracellular growth inhibition signals. To imity, the genomic structures of these genes are remarkably inter- elucidate specific targets and aberrations affecting this sub- related (Fig. 1). p14ARF and p16INK4a transcripts are produced via chromosomal region, we constructed a detailed alteration utilization of a common coding sequence for exons 2 and 3, map of the 9p21 gene cluster by analyzing homozygous together with distinct sequences for promoter and exon 1 (3). deletion, hypermethylation, and mutation of the p14ARF, However, the resulting proteins are completely different because p15INK4b, and p16INK4a genes individually in 40 esophageal different reading frames are used for the respective translation squamous cell carcinomas (ESCCs) and compared the ge- processes (4). The conservation in mammalian genome of this netic alterations with mRNA expression in 18 of these sam- unique gene structure, usually seen in primitive organisms subject ples. We detected aberrant promoter methylation of the to genome size constrains, suggests either the possible ancient p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of origin or the biological essentialness of the unitary inheritance of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methy- these two genes. p15INK4b is highly homologous to p16INK4a, lations were exclusive, whereas all but one of the p14ARF/ particularly in exon 2, where they share 91% sequence identity (5), p15INK4b methylations were accompanied by concomitant indicating their origination by a gene duplication event. The 9p21 p16INK4a methylation. We detected homozygous deletion of gene cluster is the first of its kind identified in the human genome p16INK4a in 7 (17.5%), of p14ARF-E1b in 13 (33%), and of associated with multiple tumor suppressor activities. Besides its p15INK4b in 16 (40%) tumor samples. Most deletions oc- functional importance in regulating cell proliferation, which makes curred exclusively on the E1b-p15INK4b loci. Two samples this gene cluster a target of selective inactivation during carcino- contained p14ARF deletion but with p16INK4a and p15INK4b genic process, there may be a physical basis underlining its frequent intact. No mutation was detected in the p14ARF and p16INK4a disruption in cancer. It was reported that at least two tightly clus- genes. Comparative RT-PCR showed good concordance be- tered breakpoints exist within the cluster, the sequence context of tween suppressed mRNA expression and genetic alteration which potentially facilitates illegitimate V(D)J recombinase activ- for p15INK4b and p16INK4a genes in the 18 frozen samples, ities (6). Furthermore, the promoter regions of all three genes are whereas 5 of the 13 cases with suppressed p14ARF mRNA highly abundant with CpG islands that are susceptible to hyperm- expression contained no detectable E1b alteration but aber- ethylation (7). It is intriguing why presumably important genes are clustered in such a manner that renders high susceptibility to genetic alterations. Recent studies have revealed that p14ARF and p16INK4a Received 4/26/99; revised 8/2/99; accepted 8/2/99. play active roles in the p53 and Rb growth-control pathways, The costs of publication of this article were defrayed in part by the respectively (Fig. 1; Ref. 8). p16INK4a is a cyclin-dependent payment of page charges. This article must therefore be hereby marked kinase inhibitor functioning upstream Rb. It can negatively advertisement in accordance with 18 U.S.C. Section 1734 solely to regulate cell cycle progression by preventing the phosphoryla- indicate this fact. ARF 1 Supported by NIH Grant CA65781, facilities from National Institute of tion (inactivation) of Rb during G1 phase (9). p14 restrains Environmental Health Sciences Center Grant ES 05022, National Can- cell growth by abrogating Mdm2 inhibition of the p53 activity, cer Institute Cancer Center Supporting Grant CA 72030, and National and therefore facilitates p53 mediated cell cycle arrest and Natural Science Foundation of China (39840012). apoptosis (10). It was demonstrated that oncogenic Ras elicits an 2 These authors contributed equally to this study. 3 anti-tumorigenic response mediated by the up-regulation of both To whom requests for reprints should be addressed, at Laboratory for ARF INK4a Cancer Research, College of Pharmacy, Rutgers University, Piscataway, p14 and p16 , which in turn activate the tumor suppres- NJ 08854-8020. Phone: (732) 445-5360; Fax: (732) 445-0687. sors p53 and pRb, respectively (11, 12). Recent study further Clinical Cancer Research 2705 Fig. 1 Genomic organization of the 9p21 gene cluster and schematic de- scription of the involvement of p14ARF, p15INK4b, and p16INK4a gene products in the pRb and p53 antitumorigenetic pathways [based on a diagram that ap- peared in Robertson et al. (7)]. Solid lines, regulatory steps of each pathway; dashed lines, cross-talk between the pRb and p53 pathways. showed that p14ARF provides a failsafe mechanism for defective deletion, whereas p15INK4b was frequently homozygously de- Rb pathway by its inducibility via deregulated E2F-1 activity leted and occasionally methylated (20). This pattern is different resulted from Rb inactivation (13). Compared with p14ARF and from reports on other types of cancers identifying p16INK4a as p16INK4a, p15INK4b is less prominent as a tumor suppressor. In the main deletion target and p15INK4b as a bystander, which contrast to p16INK4a, which is activated by intracellular stimuli, possibly play a small part in the tumor suppressor role (2). In p15INK4b suppresses cell growth in response to extracellular light of the recent elucidation of the tumor suppressor role of stimuli such as TGF-b (5). p14ARF, the first exon of which is only 12 kb downstream of the Inactivation of the Rb and p53 tumor suppressor pathways p15INK4b gene, and the presence of evidence of exclusive dele- is observed in most human cancers (1). By virtue of its close tion of p14ARF-E1b with the retention of both p15INK4b and involvement in both pathways, the p14ARF-p15INK4b-p16INK4a p16INK4a in T-cell acute lymphoblastic leukemia (21), we sus- gene cluster at chromosome 9p21 may be a nexus of the cellular pect that our previous result may indicate p14ARF-E1b as being growth-control network, the inactivation of which results in a primary target for inactivation in ESCCs. In consideration of collapse of the tumor suppression system. To date, a vast this point, we thought it is necessary to investigate whether amount of data has demonstrated multiple types of genetic genomic alterations indeed associate with altered gene expres- alterations on the 9p21 region, the prevalence of which varies sions in ESCCs. In the present study, we constructed a detailed with the type of tumors. For example, large homozygous dele- alteration map of the 9p21 gene cluster by analyzing the ho- tions are common in head and neck tumors, bladder carcinomas, mozygous deletion and aberrant methylation of the p14ARF, and malignant gliomas (14–16). Transcriptional-inactivating p15INK4b, and p16INK4a genes individually in 40 resected ESCC promoter methylation was common in breast and colon cancer samples and analyzed the mRNA expression pattern of the for the p16INK4a gene (17) and in leukemia for p15INK4b (17, respective genes in 18 frozen samples. We show that suppressed 18). Recently, Robertson et al. (7) identified that the promoter of mRNA expression occurred at a high frequency for all three p14ARF gene is a CpG island and observed its hypermethylation genes in primary ESCCs, and homozygous deletion is the pri- in colon cancer cell lines, which was responsible for the gene mary cause leading to such inactivation for the p14ARF and inactivation (7). Point mutation and small deletion of the p15INK4b genes, whereas aberrant methylation is the main event p16INK4a locus was common in pancreatic adenocarcinoma, underlining p16INK4a inactivation. biliary tract cancers, and hereditary melanoma (reviewed in Ref. INK4a ARF 19) and affected p16 exclusively or together with p14 Materials and Methods (4). Despite the ample evidence of the 9p21 gene cluster as a ESCC Specimen Preparation and RNA/DNA Extrac- frequent tumor target, it is not known whether all three genes in tion. Eighteen primary ESCC specimens, together with their the cluster are indiscriminately affected in a tumor and whether adjacent normal epithelial tissues, were collected from patients all of the genes are uniformly disrupted by the same mechanism. in Linzhou City (formerly Linxian) of northern China, a well- We reported previously an alteration pattern of the 9p21 recognized high-risk area for ESCC.