CHEK 2 Mutations Affecting Kinase Activity Together With Mutations in TP 53 Indicate a Functional Pathway Associated with Resistance to Epirubicin in Primary Breast Cancer

Background: Chemoresistance is the main obstacle to cure in most malignant diseases. Anthracyclines are among the main drugs used for breast cancer therapy and in many other malignant conditions. Single parameter analysis or global gene expression profiles have failed to identify mechanisms causing in vivo resistance to anthracyclines. While we previously found TP53 mutations in the L2/L3 domains to be associated with drug resistance, some tumors harboring wild-type TP53 were also therapy resistant. The aim of this study was; 1) To explore alterations in the TP53 gene with respect to resistance to a regular dose epirubicin regimen (90 mg/m every 3 week) in patients with primary, locally advanced breast cancer; 2) Identify critical mechanisms activating p53 in response to DNA damage in breast cancer; 3) Evaluate in vitro function of Chk2 and p14 proteins corresponding to identified mutations in the CHEK2 and p14 genes; and 4) Explore potential CHEK2 or p14 germline mutations with respect to family cancer incidence. Methods and Findings: Snap-frozen biopsies from 109 patients collected prior to epirubicin (as preoperative therapy were investigated for TP53, CHEK2 and p14 mutations by sequencing the coding region and p14 promoter methylations. TP53 mutastions were associated with chemoresistance, defined as progressive disease on therapy (p = 0.0358; p = 0.0136 for mutations affecting p53 loop domains L2/L3). Germline CHEK2 mutations (n = 3) were associated with therapy resistance (p = 0.0226). Combined, mutations affecting either CHEK2 or TP53 strongly predicted therapy resistance (p = 0.0101; TP53 mutations restricted to the L2/L3 domains: p = 0.0032). Two patients progressing on therapy harbored the CHEK2 mutation, Arg95Ter, completely abrogating Chk2 protein dimerization and kinase activity. One patient (Epi132) revealed family cancer occurrence resembling families harboring CHEK2 mutations in general, the other patient (epi203) was non-conclusive. No mutation or promoter hypermethylation in p14 were detected. Conclusion: This study is the first reporting an association between CHEK2 mutations and therapy resistance in human cancers and to document mutations in two genes acting direct up/down-stream to each other to cause therapy failure, emphasizing the need to investigate functional cascades in future studies. Citation: Chrisanthar R, Knappskog S, Løkkevik E, Anker G, Østenstad B, et al. (2008) CHEK2 Mutations Affecting Kinase Activity Together With Mutations in TP53 Indicate a Functional Pathway Associated with Resistance to Epirubicin in Primary Breast Cancer. PLoS ONE 3(8): e3062. doi:10.1371/journal.pone. 0003062 Editor: Toru Ouchi, Northwestern University, United States of America Received April 1, 2008; Accepted July 31, 2008; Published August 26, 2008 Copyright: 2008 Chrisanthar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Norwegian Cancer Society, the ‘‘Rosa sløyfe’’ Breast Cancer Fund Raising, the Norwegian Health Region West (HelseVest) and the Innovest program of Excellence. Ranjan Chrisanthar was a recipient of a fellowship from Helse Vest. The Clinical Trial research office is financially supported by the Norwegian Cancer Society. None of the funders had any role with respect to study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: per.lonning@helse-bergen.no PLoS ONE | www.plosone. org 1 August 2008 | Volume 3 | Issue 8 | e3062