Abstract 486: The novel tumor suppressorDIAPH3governs transition to an amoeboid phenotype

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: We previously reported that loss of DIAPH3, proximal to RB1 on chromosome 13q and encoding the cytoskeletal regulator Diaphanous-related Formin-3 (DRF3), is associated with metastatic prostate cancer. Here we examine further the impact of genomic DIAPH3 loss on motility and metastasis, in multiple cancers. Methods: We employed publically available genomic data sets (including the Genomic Identification of Significant Targets in Cancer platform), Oncomine expression profiles, Affymetrix Human Genome-wide SNP arrays, and tissue microarrays to determine DIAPH3 copy number and DRF3 expression in carcinomas and with disease progression. In cultured cancer cells, biochemical and cell biological approaches in two- and three-dimensional growth conditions were used to assess mechanisms underlying phenotypic changes associated with DRF3 deficiency. The in vivo impact of DIAPH3 loss was investigated with a murine experimental metastasis model. Results: Analysis of somatic copy number alterations along chromosome 13q revealed a region of significant focal deletion, in which the DIAPH3 locus was central, common to hepatocellular, breast, and prostate carcinomas. Employing prostate cancer as a disease-relevant model, we detected reduced DIAPH3 copy number in multiple, independent patient cohorts. DIAPH3 deletions accumulated during disease progression, were strongly associated with metastatic disease, and were prevalent in disseminated tumor cells from patient bone marrow aspirates. Tissue microarrays confirmed reduced expression of DRF3 in metastases relative to normal tissue and primary tumor. In vitro, stable silencing of DRF3 in prostate cancer and HRAS-transformed human mammary epithelial cells dramatically altered cell morphology, evoking an ‘amoeboid’ morphological phenotype. Accordingly, biochemical hallmarks of amoeboid behavior, as well as enhanced migration, invasion, and chemotaxis, were associated with DRF3 loss. Consistent with its role as a regulator of cytoskeletal dynamics, DRF3 depletion destabilized microtubules, thereby perturbing endocytic receptor trafficking. EGFR was observed to accumulate in endosomes and was hyperactive, coinciding with persistent signaling through the MEK-ERK pathway. Inhibition of MEK reversed the amoeboid phenotype stimulated by DRF3 loss. Enforced expression of DRF3 accelerated down-regulation of EGFR and suppressed amoeboid morphology. Phosphorylation was observed to modulate DRF3 function. In a murine metastasis model, the formation of pulmonary lesions was markedly potentiated by DRF3 silencing, supporting a role for the protein in mitigation of tumor cell dissemination. Conclusions: Genomic deletions at the DIAPH3 locus are prevalent in multiple carcinomas and are strongly associated with metastasis. DRF3 is a metastasis suppressor that prevents transition to an amoeboid tumor cell phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 486. doi:1538-7445.AM2012-486