Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers

Abstract

PolyA simple repeat sequence deletions are common in tumors with microsatellite instability (MSI+). Such deletions occur one base at a time in DNA mismatch repair (MMR)-deficient yeast suggesting larger deletions in human MSI+ tumors represent multiple sequential stepwise losses. Sum total deletions in four polyA repeats were variable (between -17 to -45 bp) in 20 sporadic MSI+ colorectal cancers. Progressive but less extensive total deletions (maximum of -12 bp) occurred in similar polyA sequences in MMR-deficient mice (mlh1-/-) up to 478 days old. PolyA repeat lengths were relatively stable but already shortened in the MMR-deficient cell line HCT116. A transgene with 26 A's transfected into HCT116 shortened an average of 3.8 bases pairs after 469 days in culture, less than average deletions of BAT25 (-5.3) or BAT26 (-9.0) in MSI+ cancers. These findings further suggest that extensive polyA deletions common in MSI+ tumors likely reflect multiple stepwise smaller deletions that accumulate more than hundreds of divisions after loss of MMR.

Figure 1.

A: Examples of BAT26 and BAT40 deletions in MSI+ colorectal cancers. Lengths of the alleles are estimated from the densest band (open circle, normal; filled circle, tumor). B: Total deletions in BAT20, 25, 26, and 40 were variable for the 20 MSI+ colorectal cancers (specimens 1 to 20). For comparison, estimated total deletions for MMR-deficient (specimens 21 to 23; HCT116, SW48, HCT15) and MMR-proficient (specimens 24 to 26; HT29, SW480, SW837) colorectal cancer cell lines are illustrated. Deletions for the cell lines are estimates because matching normal tissues were unavailable and their starting germline sizes were assumed to be equal to the average lengths found in the 20 MSI+ tumor patients. C: Normal (open circles) and tumor (filled circles) allele lengths for MSI+ cancers and cell lines (triangles) at the four polyA loci.

Figure 2.

A: Serial measurements of BAT loci [BAT20 (X), 25 (filled circle), 26 (open circle), and 40 (triangle)] in a clonal isolate of HCT116. Further deletions in the already shortened BAT alleles were rare during 352 days in culture. B: Serial measurements of mbat loci (Table 1) ▶ in large and small intestines of mlh1−/− mice. In contrast to HCT116, progressive deletions occurred in these longer polyA repeats. Deletions in the oldest mouse (478 days old) were less than the MSI+ cancers (Figure 1) ▶ .

Figure 3.

Distribution of polyA repeat lengths in MSI+ cancers (BAT25, shaded bars; BAT26, black bars) and the A26 transgene in HCT116 (open bars). Deletions in the A26 transgene were variable and less extensive than BAT loci in MSI+ cancers after 469 culture days. Arrows indicate average deletions.