Abstract P5-03-01: Focal adhesion kinase inhibition abrogates ductal carcinoma in situ cancer stem cell activity and self-renewal capacity via down regulation of WNT3a and potentiates effects of radiation

Abstract

Abstract Background - Ductal carcinoma in situ (DCIS) recurs in 20% of patients after conserving surgery and radiotherapy. Research shows cancer stem cells (CSCs) are tumour initiating, resistant to current therapies and therefore may play a role in cancer recurrence. Focal Adhesion Kinase (FAK) signalling plays an important role in breast cancer, metastasis and radioresistance. These studies investigate FAK signalling in the regulation of human DCIS CSCs. Methods - Immunohistochemical (IHC) expression of active FAK was analysed in DCIS patients (n = 63) with known recurrence. DCIS cell lines (DCIS.com & SUM225) and human primary DCIS cells (n = 13) were cultured as primary and secondary mammospheres to assess CSC/progenitor activity and self-renewal after inhibition of FAK (PF573228 or siRNA) in combination with radiation (2Gy). Western Blotting and IHC staining were used to measure DNA-damage, FAK and Wnt signalling. Cell lines were cultured in 3D matrigel as DCIS acini and in vivo as DCIS xenografts to examine the effects of FAK inhibition. Results - Mammosphere initiating cells from DCIS cell lines and human primary DCIS have increased FAK activity and are more radioresistant than the total population (TP) (TP-6Gy vs AR-6Gy; DCIS.com-0.51±0.01 vs 0.05±0.01 Survival fraction (SF), P<0.001; SUM225-0.52±0.03 vs 0.03±0.01 SF, P<0.001; patient-derived DCIS–67.61±6.4% vs 13.8±7% mammosphere formation efficiency (MFE), P = 0.0005). FAK inhibition using PF573228 and FAK siRNA in mammosphere or 3D matrigel culture reduced mammosphere formation alone (mammosphere culture-control vs PF573228 0.5μM; DCIS.com-1.2±0.09% vs 0.7±0.06% MFE, P<0.001; SUM225-2.2±0.1% vs 1.4±0.06% MFE, P<0.001; patient derived DCIS-100±2.6% vs 72.1±6.2% MFE compared to control, P<0.001). Moreover, FAK inhibition in combination with 2Gy radiation abrogated mammosphere formation more than either treatment alone and increased DNA double strand breaks (2Gy vs 2Gy/0.5μM PF573228; DCIS.com-0.66±0.02% vs 0.39±0.01% MFE, P<0.05; SUM225-1.59±0.06% vs 1.22±0.05% MFE, P<0.01; patient derived DCIS-0.63±0.04 vs 0.39±0.03 fold change compared to control, P<0.01). Expression of active FAK in DCIS (n = 63) was associated with a shorter time to recurrence after surgery (Univariate analysis Cox's regression model, p = 0.049). Further investigation revealed FAK inhibition also decreased self-renewal capacity (SR) in vitro and in vivo (control vs PF573228 0.5μM; DCIS.com-0.85±0.03 vs 0.4±0.08 SR, P<0.001; SUM225-0.66±0.06 vs 0.28±0.03 SR, P<0.001; patient-derived DCIS-1 vs 0.54±0.19 SR compared to control, P = 0.001; DCIS.com xenograft-vehicle vs PF562271 25mg/kg; 1.3±0.12% vs 0.65±0.05% MFE, P<0.0001) and could be rescued through the addition of Wnt3a, showing for the first time FAK-Wnt signalling crosstalk in DCIS. Conclusion - Targeting FAK reduced self-renewal of DCIS stem/progenitor cells via down regulation of Wnt3a and improves sensitivity to radiotherapy. These data would support further investigations through a peri-operative trial of short term FAK inhibition before radiotherapy on the recurrence rates of DCIS patients after breast conserving surgery. Funding - Breast Cancer Campaign and The Royal College of Surgeons. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-01.