Dong K. Chang

The biochemical basis of microsatellite instability and abnormal immunohistochemistry and clinical behavior in Lynch Syndrome: from bench to bedside

Lynch syndrome is an inherited disease caused by a germline mutation in one of four DNA mismatch repair (MMR) genes. The clinical manifestations can be somewhat variable depending upon which gene is involved, and where the mutation occurs. Moreover, the approach to the diagnosis of Lynch syndrome is becoming more complex as more is learned about the disease, and one needs to understand how the DNA MMR proteins function, and what makes them malfunction, to have an optimal appreciation of how to interpret diagnostic studies such as microsatellite instability and immunohistochemistry of the DNA MMR proteins. Finally, an understanding of the role of the DNA MMR system in regulation of the cell cycle and the response to DNA damage helps illuminate the differences in natural history and response to chemotherapeutic agents seen in Lynch syndrome.

Mad-1 Is the Exclusive JC Virus Strain Present in the Human Colon, and Its Transcriptional Control Region Has a Deleted 98-Base-Pair Sequence in Colon Cancer Tissues

ABSTRACT JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23- and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [χ2 = 23.6;P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.

EPIGENETIC INACTIVATION OF RUNX3 IN MICROSATELLITE UNSTABLE SPORADIC COLON CANCERS

Runt domain transcription factors are important targets of TGF‐β superfamily proteins and play a crucial role in mammalian development. Three mammalian runt‐related genes, RUNX1, RUNX2 and RUNX3, have been described. RUNX3 has been shown to be a putative tumor suppressor gene localized to chromosome 1p36, a region showing frequent loss of heterozygosity events in colon, gastric, breast and ovarian cancers. Because of the important role of TGF‐β signaling in the human colon, we hypothesized that RUNX3 may serve as a key tumor suppressor in human colon cancers and colon cancer‐derived cell lines. We examined RUNX3 expression and the frequency of RUNX3 promoter hypermethylation in 17 colon cancer cell lines and 91 sporadic colorectal cancers. Semiquantitative analysis of RUNX3 transcripts was performed by RT‐PCR and de novo methylation of the RUNX3 promoter was studied by a methylation‐specific PCR (MSP) assay. Nineteen of 91 informative tumors (21%) and 11 of 17 (65%) colon cancer cell lines exhibited hypermethylation of the RUNX3 promoter. Interestingly, RUNX3 promoter hypermethylation was more common in tumors exhibiting high frequency of microsatellite instability (MSI‐H) (33% of MSI‐H vs. 12% of MSI‐L/MSS tumors; p = 0.012). Hypermethylation of the RUNX3 promoter correlated with loss of mRNA transcripts in all cell lines. RUNX3 promoter methylation was reversed and its expression restored in SW48 and HCT15 colon cancer cells after treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine, indicating that loss of expression is caused by epigenetic inactivation in colon carcinogenesis. This is the first demonstration of frequent de novo hypermethylation of the RUNX3 promoter in sporadic colon cancers. The significant association of RUNX3 promoter hypermethylation with MSI‐H colon cancers suggests that RUNX3 is a novel target of methylation, along with the hMLH1 gene, in the evolution of MSI‐H colorectal cancers.

Detection of JCV DNA sequences in colon cancer cell lines by fluorescent in situ hybridization

The inhibition of apoptosis is a crucial early step in the development of colorectal cancer (CRC), although the mechanisms are incompletely understood. In a variety of malignancies (e.g. pancreatic) there is an overexpression of the novel proto-oncogene, Akt (protein kinase B), an antiapoptotic serine-threonine kinase also implicated in Wnt signaling. Since the role of Akt in CRC has not been previously explored, we examined this by immunohistochemistry (lHC) in sporadic human adenomas and CRe. To investigate the microsatellite instability (MSI) pathway, tumors from patients with hereditary nonpolyposis colon cancer (HNPCC) were assessed. Finally, Akt was evaluated in experimental CRC using the azoxymethane (AOM)-rat model. METHODS: IHC was performed on paraffin-embedded samples with a monoclonal and polyclonal antibody for Aktl/2 using the Vectastain ABC kit. Human and animal colon tumors were selected from archived materials. Akt staining of sections was assessed by a pathologist using appropriate negative and positive controls. RESULTS and CONCLUSIONS: As demonstrated by the Table, Akt is overexpressed in approximately 22-42% of human and experimental CRe. The genetic mechanisms of tumor development (adenomatous polyposis coli mutation in sporadic CRC, f3-catenin mutations in AOM-tumors or MSI in HNPCC tumors) did not appear to significantly affect expression of this epigenetic protein. The observation that Akt overexpression occurs early in carcinogenesis (adenomas) emphasizes its biological significance. Our recent demonstration that Akt downregulation is important in nonsteroidal antiinflammatory drugs (NSAID) induction of apoptosis is supported by the observation that AOM-tumors from animals that had been treated with NSAIDS had a trend towards lower Akt expression than those on a control diet (60 versus 28 % although the numbers were small). This data suggests that Akt overexpression is important in the early dysregulation of apoptosis and Wnt signaling which characterizes CRC development. GASTROENTEROLOGY Vol. 118, No.4

JC virus DNA sequences are frequently present in the human upper and lower gastrointestinal tract

BACKGROUND & AIMS JC virus (JCV), a human polyomavirus, has been found in a limited number of normal human tissues and cancers. The oncogenic potential of this virus is mediated by a transforming protein, the T antigen (TAg). We have previously demonstrated the presence of JCV-TAg in colorectal cancers, in adjacent normal colonic mucosa from these patients, and in the human colon cancer cell line SW480. The mode of transmission of this virus is unclear, and we hypothesized that the gastrointestinal (GI) tract may be a reservoir for the virus. METHODS DNA was extracted from 129 normal GI tissue samples collected from 33 patients. Topoisomerase I-assisted polymerase chain reaction (PCR) was used to detect the virus using exact and degenerate primers. Nested PCR and Southern blot analysis confirmed the identity of the PCR products. Single-stranded conformation polymorphism (SSCP) analysis and sequencing were used to evaluate the presence of viral quasispecies. RESULTS JCV sequences were found in 75.8% of patients (70.6% of upper GI and 81.2% of colonic samples); no significant differences in rates of infection were found by site. The use of degenerate primers combined with topoisomerase I treatment led to viral detection in 58.9% of samples, compared with 27.9% of samples using exact primers and topoisomerase I (P < 0.01). SSCP and sequencing analysis confirmed the amplification of viral quasispecies and the authenticity of TAg sequences. CONCLUSIONS The results show that JCV DNA sequences are highly prevalent in the human upper and lower gastrointestinal tract of immunocompetent individuals.