Abstract P1-05-13: CLIC3 is associated with invasive behaviour and poorer prognosis in estrogen receptor-negative breast cancer.

Abstract

Background: We previously described a Chloride Intracellular Channel-3 (CLIC3)-dependent recycling pathway which trafficked active integrins from late endosomes to the cell surface and which was required for the migratory and invasive behaviour of A2780 ovarian cancer cells in vitro. We also demonstrated that elevated expression of CLIC3 was associated with poor prognosis in pancreatic cancer. We therefore set out to investigate the role of CLIC3 in breast cancer. Materials and Methods: CLIC3 expression was assessed by immunohistochemistry and the weighted histoscore method in a tissue microarray (TMA) consisting of triplicate cores from 141 patients diagnosed with invasive estrogen receptor (ER)-negative early breast carcinoma between 1995 and 1998. Full clinicopathological and follow-up data were available. Further data were obtained from publicly available gene expression datasets (Desmedt, GSE7390) and Oncomine™. Knockdown of CLIC3 in the ER-ve MDA-MB231 breast cancer cell line was achieved by nucleofection of 2 different siRNA sequences (Dharmacon). Inverse invasion assays measured invasion into a plug of matrigel supplemented with fibronectin over 72 hours whilst organotypic invasion assays measured invasion into a fibroblast-containing collagen matrix over 6 days. Results: CLIC3 mRNA expression was significantly elevated in breast cancer in comparison to normal breast tissue in two independent data sets. High CLIC3 protein levels were associated with significantly shorter breast cancer specific survival (p = 0.026) in a TMA of 141 ER-ve patients. This finding was corroborated by the observation that high CLIC3 mRNA levels were associated with shorter overall survival in 198 patients in the Desmedt dataset (p = 0.038). Transient silencing of CLIC3 expression using two independent siRNA sequences had no effect on proliferation of MDA-MB231 cells but significantly reduced their invasiveness by 46% and 93% respectively in an inverse invasion assay and by 36% and 42% respectively in an organotypic invasion assay. CLIC3 was found to co-localise with Rab7 and LAMP1 in late endosomes/lysosomes in MDA-MB231 cells. Conclusions: Our clinical and in vitro data indicate an important role for CLIC3 in the invasive and metastatic behaviour of some breast cancers. Further work to elucidate molecular mechanisms is underway and will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-13.