The mutational profile and infiltration pattern of murine MLH1-/- tumors: concurrences, disparities and cell line establishment for functional analysis

Claudia Maletzki,Ernst Klar,Franziska Beyrich,Michael Linnebacher,Maja Hühns

doi:10.18632/oncotarget.10677

Claudia Maletzki, Ernst Klar + Show 3 more

Open Access

https://doi.org/10.18632/oncotarget.10677

Copy DOI

Journal: Oncotarget	Publication Date: Jul 18, 2016
Citations: 13	License type: cc-by

Affiliation: University of Rostock

Abstract

Mice lines homozygous negative for one of the four DNA mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6) were generated as models for MMR deficient (MMR-D) diseases. Clinically, hereditary forms of MMR-D include Lynch syndrome (characterized by a germline MMR gene defect) and constitutional MMR-D, the biallelic form. MMR-D knockout mice may be representative for both diseases. Here, we aimed at characterizing the MLH1-/- model focusing on tumor-immune microenvironment and identification of coding microsatellite mutations in lymphomas and gastrointestinal tumors (GIT).All tumors showed microsatellite instability (MSI) in non-coding mononucleotide markers. Mutational profiling of 26 coding loci in MSI+ GIT and lymphomas revealed instability in half of the microsatellites, two of them (Rfc3 and Rasal2) shared between both entities. MLH1-/- tumors of both entities displayed a similar phenotype (high CD71, FasL, PD-L1 and CTLA-4 expression). Additional immunofluorescence verified the tumors’ natural immunosuppressive character (marked CD11b/CD200R infiltration). Vice versa, CD3+ T cells as well as immune checkpoints molecules were detectable, indicative for an active immune microenvironment. For functional analysis, a permanent cell line from an MLH1-/- GIT was established. The newly developed MLH1-/- A7450 cells exhibit stable in vitro growth, strong invasive potential and heterogeneous drug response. Moreover, four additional MSI target genes (Nktr1, C8a, Taf1b, and Lig4) not recognized in the primary were identified in this cell line.Summing up, molecular and immunological mechanisms of MLH1-/- driven carcinogenesis correlate well with clinical features of MMR-D. MLH1-/- knockout mice combine characteristics of Lynch syndrome and constitutional MMR-D, making them suitable models for preclinical research aiming at MMR-D related diseases.

Highlights

Lawrence Loeb who described possible relations hips between DNA replication errors and malignant progression in 1974 initially formulated microsatellite instability (MSI) or mismatch repair deficiency (MMR-D) as a mutator phenotype [1]
Lymphomas were detected in the thymus (n=3), spleen (n=7), liver (n=5), kidney (n=3), and duodenum (n=2)
Due to the higher aggressiveness of these hematological malignancies, lymphoma development was usually seen before mice were 40 weeks old, while gastrointestinal tumors were only seen at later time (> 42 weeks), which is consistent with the literature [6]

Summary

Introduction

Lawrence Loeb who described possible relations hips between DNA replication errors and malignant progression in 1974 initially formulated microsatellite instability (MSI) or mismatch repair deficiency (MMR-D) as a mutator phenotype [1]. As a consequence of MMR-D, unrepaired mutations become scattered throughout the genome, this situation defines the mutator or MSI phenotype and is present in virtually all LS-associated cancers. A unique characteristic of MSI+ cancers is the expression of neoantigens due to frameshift mutations (FSM; often 1 or 2 bp deletions) in coding regions of genes. Such FSM constitute true targets for and may contribute to MMR-D-driven mutagenesis and have recently been recognized as ideal (immunological) antigens for vaccination strategies [5]

Methods

Results

Conclusion