Eric Poe Xing

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.

p16INK4a and p15INK4b genes, which encode two functionally related CDK inhibitors, recently emerged as candidate tumor suppressor genes since they were both localized to 9p21, which frequently undergoes hemizygous and homozygous deletion in a variety of tumor types. To determine the mode of inactivation of these two genes in human esophageal squamous cell carcinoma (ESCC), we performed multiple molecular analyses in 60 ESCC specimens from Linxian, China using DNA methylation assay, LOH analysis, deletion screening and SSCP-sequencing. We observed that p16INK4a inactivation was predominantly associated with aberrant methylation in the CpG island of its promoter region, whereas p15INK4b frequently had homozygous deletions. Compared with aberrant methylation, which occurred in 17 of 34 cases, homozygous deletion of p16INK4a and LOH at its nearby D9S942 microsatellite marker were observed at a much lower frequency (17%). Intragenic mutation in p16INK4a gene was rare. In contrast, homozygous deletion in p15INK4b and LOH at the nearby D9S171 marker were observed at frequencies of 35 and 47%, respectively, and the two events were significantly associated with each other. On the other hand, aberrant methylation of p15INK4b was relatively infrequent (6/34) and occurred concomitantly with p16INK4a methylation. Among the 60 cases, only four contained a continuous homozygous deletion spanning both p15INK4b and p16INK4a. Six cases were exclusively deleted at p16INK4a and 17 exclusively deleted at p15INK4b. LOH at D9S942 and D9S171 was also found to be mutually exclusive. Our results suggest that the alteration mode at 9p21 was not uniform, and the two genes were inactivated by distinct mechanisms. Altogether, 68% of the samples harbor at least one type of alteration in p16INK4a gene and 50% of the samples were altered in p15INK4b gene, indicating that they are the frequent inactivating targets during ESCC development.

To understand the alterations of Rb tumor suppressor gene and the relationship between defects in the Rb and p53 pathways in human esophageal carcinogenesis, we examined the loss of heterozygosity (LOH) of the Rb gene and immunohistochemical staining of pRb protein in 56 esophageal squamous cell carcinoma specimens and related the results to the p53 gene alterations. Using four introgenic polymorphic markers as probes, we observed LOH of the Rb gene in 30 of the 55 informative tumor samples. Immunohistochemical analysis revealed different patterns of pRb expression among the tumor samples. In the 56 cases, 16 displayed extensive pRb staining comparable to that of the adjacent normal epithelia, whereas 33 showed either significantly decreased or no pRb staining and 7 had a focal staining pattern reflecting heterogeneous cancer nests in the tumor with respect to Rb status. In the tumor samples containing Rb LOH, 90% showed low or no pRb expression, whereas in samples without Rb LOH, only 20% had altered pRb expression. There was a strong association between LOH of the Rb gene and alteration of pRb expression in our samples (P < 0.0001), suggesting LOH is a main event leading to Rb inactivation. We found that Rb LOH was more frequent in tumors with p53 mutations (P < 0.05), which occurred in 31 of the 49 cases analyzed. When the status of Rb and p53 alterations was evaluated by the combined results of immunohistochemical and genetic analyses, we found that alteration of Rb and p53 had an even stronger association in our esophageal squamous cell carcinoma samples (P = 0.0015). Among the 51 cases in which both the Rb and p53 status were determined, 31 contained alterations in both genes, and only 5 and 6 cases were altered in only Rb and only p53, respectively. Our results suggest that defects in the Rb and p53 pathways and their potential synergistic effect in deregulating cell cycle and apoptosis are major mechanisms for esophageal carcinogenesis.

Alteration of p19 mRNA expression in esophageal cancer tissue from patients at high incidence area in northern China