Abstract P2-06-07: Investigating the role of focal adhesion kinase in regulating CSC activity in invasive ductal carcinoma
Authors: Timbrell S , Aglan H , Cramer A , Foden P , Weaver D , Pachter J , Farnie G , Clarke R , Bundred N 
Published: May 2019

Background

Breast Cancer Stem-like Cells (BCSCs) have been associated with tumour development, metastasis and recurrence1. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase classically known for its role in metastasis, proliferation and survival. We have previously shown FAK plays a role in regulating CSC activity in DCIS2. We aimed to investigate FAK and CSC marker expression in a retrospective patient cohort. We aimed to evaluate the effects of FAK inhibition on CSC activity in Invasive Ductal Carcinoma.

Methods

Using a retrospective case-control cohort of 244 patients across a range of molecular phenotypes we evaluated FAK Immunohistochemical expression alongside CSC markers; Aldehyde Dehydrogenase 1 (ALDH1) and Integrin Alpha 6 (ITGa6). FAK expression was measured in IDC cell lines and ALDEFLUOR high expressing cells. FAK was inhibited using 0.5μM VS4718 or SiRNA and CSC activity evaluated in 5 cell lines and 25 patient samples. We determined the effects of 50mg/kg VS4718 for 4 weeks as single agent or in combination with Paclitaxel 7.5mg/kg in a ER-/PR-/HER- Patient Derived Xenograft model (PDX).

Results

Total FAK expression was associated with reduced breast cancer survival. Co-expression of FAK and either BCSC marker was associated with the poorest survival.

FAK and CSC marker expression pFAKtFAKALDH1ITGα6tFAK and ALDH1tFAK and ITGα6Recurrence Risk0.58 (0.31-1.08) p = 0.0842.05 (1.23-3.43) p = 0.0062.21 (1.20-4.05) p=0.0111.54 (0.92-2.23) p=0.107  Breast Cancer Death0.41 (0.12-1.51) p=0.1824.84 (2.33 -10.04) p = &lt;0.0016.58 (1.87-23.10) p=0.0032.23 (1.08-4.58) p=0.03016.7 (3.7-73.9) p=&lt;0.00112.8 (1.37-13.2,) p=0.012Hazard ratios calculated using cox-proportional hazard regression analysis.

pFAK was higher in ALDEFLUOR expressing cells and triple negative cell lines. SiRNA knockout of FAK reduced mammosphere formation, self-renewal and ALDEFLUOR expression from 1.2% to 0.2% (p=&lt;0.01, unpaired t-test) in MDA-MB-231 cells. VS4718 reduced primary mammosphere forming efficiency in all cell lines and reduced self-renewal in ER negative cell lines. FAK inhibition led to a reduction in mammosphere forming efficiency and self-renewal in 25 primary breast cancer specimens as outlined below:

FAK inhibition reduces MFE Primary Breast Cancer samples  ER negative cell lines   ER+/PR+/ Her2-ER-/PR-/Her2+ER-/PR-/Her2-SKBr3MDA-MB-231SUM159Primary mammosphere formation51.1% (n=17)53.2% (n=4)49.6% (n=5)74.1%84.8%67.6%Secondary mammosphere formation45.8% (n=8) 43.9% (n=3)42.1%57.9%47.5%Percentage of mammospheres formed given relative to control. Cell line work, minimum n=6. All above significant p=&lt;0.05, One way Anova used with post hoc Tukeys test.

VS4718 reduced tumour growth, Ki67 staining and CSC activity in our triple negative PDX model. VS4718 administration reduced ex-vivo mammosphere formation, tumour initiating capacity and prevented ALDEFLUOR enrichment when used in combination with Paclitaxel.

Conclusions

FAK, ALDH1 and ITGa6 are associated with increased breast cancer mortality in early breast cancer. Inhibition of FAK reduces CSC activity in vitro and in vivo in cell lines and patient samples. This data suggest that FAK inhibition may be used to reduce CSC activity in triple negative carcinoma.

1. Williams et al, Stem cells 2015.

Citation Format: Timbrell S, Aglan H, Cramer A, Foden P, Weaver D, Pachter J, Farnie G, Clarke R, Bundred N. Investigating the role of focal adhesion kinase in regulating CSC activity in invasive ductal carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-07.