P1-04-04: Focal Adhesion Kinase Plays a Major Role in the Regulation of Human Ductal Carcinoma In Situ (DCIS) Stem Cell Activity.

Abstract

Abstract Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that has been implicated in the regulation of normal mammary stem cells and cancer stem cells (CSCs) within mouse models. FAK is frequently overexpressed in human breast cancers but its role in human CSCs is not known. We aimed to investigate the role of FAK in human ductal carcinoma in situ (DCIS) stem cells (SCs). We have used PF573228 to inhibit FAK activity in SUM225 (HER2−overexpressing) and MCF10DCIS.com (HER2−normal) DCIS cell lines. PF573228 inhibits the autophosphorylation of FAK at tyrosine 397 causing inactivation. Western blot analysis confirmed PF573228 (0.1-5 μM) caused a dose dependent inhibition of FAK activity in both cell lines. The in vitro mammosphere colony assay was used to measure CSC activity in both DCIS cell lines and primary DCIS samples (n=5) (LREC#01/012). Mammosphere-forming efficiency (MFE) was calculated as percentage colony formation (>60μm) in the presence or absence of PF573228 (0.1-5μM). MFE was reduced in both the SUM225 cell line (p=0.02) and in the MCF10DCIS.com cell line (p=0.04) by PF573228 at a dose concentration of ≥0.1 μM (see Table 1). PF573228 (1 μM) also reduced mammosphere formation in human primary DCIS cells (n=5) by 18.1±5.75% when compared to control (p=0.03). To measure the effect of PF573228 on DCIS SC self-renewal capacity we performed secondary generation mammosphere colony assays. Primary generation mammospheres that had been treated with PF573228 (0.1-5μM) were dissociated into a single cell suspension and reseeded out into mammosphere culture conditions with no additional PF573228. The mammosphere regeneration ratio (MRR) was calculated as the proportion of secondary mammospheres formed relative to the number of primary mammospheres. MRR was reduced in both the SUM225 (p=0.005) and MCF10DCIS. com (p=0.001) cell lines after treatment with ≥0.5μM or ≥1.0μM PF573228 respectively (see Table 2). Preliminary data in human primary DCIS cells (n=1) also shows a reduction in MRR, from 0.56 to 0.14 with 0.5μM PF573228. These results suggest that CSC self-renewal capacity is reduced by FAK inhibition. We conclude that FAK signalling is important in human DCIS SC activity measured by mammosphere growth and regeneration. Targeting FAK in the treatment of DCIS may reduce disease recurrence and improve patient outcome. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-04.