Somatic mutation of Afadin leads to anchorage independent survival and metastatic growth of breast cancer through alpha E-catenin dependent destabilization of the adherens junction

Abstract

Loss of E-cadherin (CDH1) and the adherens junction (AJ) drive development and progression of invasive lobular breast cancer (ILC). However, approximately 40% retain wild type CDH1 alleles, indicating that modulation of other genes attenuates the AJ during ILC etiology. To identify alternative drivers, we performed targeted sequencing in CDH1 wild type samples, based on a defined set of 100 AJ, tight junction, and desmosome genes we designated as the Adhesome. In 146 ILC samples, we identified 62 cases (43%) with wild type CDH1 alleles in which we detected a total of 284 mutations in 36 Adhesome genes. After selection based on occurrence and potential loss of function, we identified an inactivating frameshift mutation in Afadin (AFDN; p.Lys630fs). Functional studies in E-cadherin-expressing breast cancer cells showed that Afadin knockout leads to immature AJs, and a non-cohesive phenotype accompanied by actomyosin dependent anoikis resistance, which are classical ILC hallmarks. Afadin reconstitutions show that F-actin organization critically depends on the E-catenin binding CC domain. Afadin loss in intraductal xenograft mouse breast cancer models leads to ILC-type morphologies and overt lung metastases. AFDN truncate reconstitutions revealed that deletion of the C-terminal E-catenin binding CC domain is sufficient to drive metastatic ILC. In conclusion, we identified and functionally coupled a somatic frameshift AFDN mutation in breast cancer to destabilization the epithelial AJ and the development of ILC hallmarks such as actomyosin-dependent anoikis resistance and single cell invasion. As such, Afadin represents a candidate tumor suppressor for E-cadherin-positive ILC development and progression.