Tumor-associated Apc mutations in Mlh1−/−Apc1638N mice reveal a mutational signature of Mlh1 deficiency

Abstract

Apc 1638N mice, which are heterozygous for a germline mutation in Apc, typically develop three to ®ve spontaneous intestinal tumors per animal. In most cases this is associated with allelic loss of wildtype Apc. We have previously reported that the multiplicity of intestinal tumors is increased dramatically by crossing Apc 1638N with an Mlh1-de®cient mouse strain that represents an animal model of hereditary non-polyposis colorectal cancer (HNPCC). The increased tumor multiplicity in these mice was associated with somatic mutations in the Apc tumor suppressor gene. Here, we have examined the nature and distribution of 91 Apc mutations implicated in the development of intestinal tumors in Mlh1 7/7 Apc 1638N animals. Protein truncation mutations were detected in a majority of tumor samples, indicating that the prevailing mechanism of Apc mutation in tumors is altered from allelic loss to intragenic mutation as a result of Mlh1 de®ciency. The observed mutations were a mixture of base substitutions (27%) and frameshifts (73%). Most frameshifts were detected within dinucleotide repeats and there were prominent mutational hotspots within sequences of this sort at codons 927 ± 929, 1209 ± 1211 and 1461 ± 1464. The observed Apc mutations caused protein truncation upstream of the third 20 amino acid b-catenin binding domain and the ®rst Axin-binding SAMP repeat, yielding Apc proteins that are predicted to be de®cient in destabilizing b-catenin. Our results reveal a characteristic mutational signature in Apc that is attributable to Mlh1 de®ciency. This demonstrates a direct e ect of Mlh1 de®ciency in the mutation of Apc in these tumors, and provides data that clarify the role of Mlh1 in mammalian DNA mismatch repair.

Contributions by MutL Homologues Mlh3 and Pms2 to DNA Mismatch Repair and Tumor Suppression in the Mouse

“…Kaplan-Meier survival curves were generated as previously done (10,(22)(23)(24)(25)(26)(27). The numbers of mice included in survival analyses are Wt (n = 34), Mlh3 +/À (n = 27),…”

“…Statistical significance between genotypes was determined using the log-rank test as previously done (10,(22)(23)(24)(25)(26)(27). Analysis of tumors.…”

Haploinsufficiency of Flap endonuclease ( Fen1 ) leads to rapid tumor progression

“…Codons 677-1674 of the mouse Apc gene were analyzed for protein truncating mutations by PCR and in vitro transcription and translation (IVTT) (51)(52). PCR amplification of the WT Apc allele was performed in two stages to eliminate coamplification of the inactivated Apc 1638N allele.…”

Somatic mutations of APC gene in carcinomas from hereditary non-polyposis colorectal cancer patients

“…Animal studies further supported the notion that APC mutations in Msh6(-/-)Apc1638N mice consisted predominantly of base substitutions (93%) creating stop codons, however, in Msh3(-/-)Msh6(-/-)Apc1638N tumors, a mixture of base substitutions (46%) and frameshifts (54%) was observed, indicating that Msh3 could suppress frameshift mutations of Apc gene in Msh6(-/-)Apc1638N mice [25] . The observation demonstrated that type of APC mutation was closely related Huang J et al Exploring the possible arising mechanism of somatic APC mutation and its roleto the function of specific MMR gene while in Mlh1-/-Apc1638N animals, the prevailing mechanism of APC mutation in tumors was altered from allelic loss to intragenic mutation as a result of Mlh1 deficiency, the observed mutations were more frameshifts (73%) than base substitutions (27%), most frameshifts were located within dinucleotide repeats [26] . We therefore hypothesized that defective MMR might precede APC mutation in some HNPCC cases whereas for those frameshifts not showing microsatellite alterations, the initiation mechanism for these tumors was not clear yet and proposed to be similar to that for sporadic CRCs [2,3,5,12] , which meant that APC mutation, rather than genomic instability, might be the initiating event in tumorigenesis.…”