Abstract A70: The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma
Authors: Begum Asma , Ewachiw Theodore , McMillan Ross , Penchev Vesselin , Jung Clinton , Huang Ally , Norberg Jessica , Rajeshkumar NV , Maitra Anirban , Matsui William , Rasheed Zeshaan A. 
Published: October 2015

Introduction: Despite recent advancements in treatment, pancreatic ductal adenocarcinoma (PDAC) continues to carry a very poor prognosis, which is largely attributable to drug resistance and early metastasis. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. In order to better understand the mechanisms that regulate PDAC CSC maintenance and function, we have focused on their interactions with the extracellular matrix (ECM), a major component of the tumor microenvironment in PDAC.

Methods: We used PDAC cell lines (Capan-1, MIA PaCa-2, and BxPC-3) and low passage human xenografts to evaluate the effects of a panel of ECM proteins (type I collagen, type IV collagen, fibronectin, and laminin) on CSC function and content. PDAC cells were plated on ECM protein coated plates (Fisher Scientific) and harvested at 72-96 hrs. We plated cells in 3% methylcellulose to assess clonogenic growth and on 8-µm transwell filters (Corning) to assess cell motility. We analyzed ECM-mediated activation of focal adhesion kinase (FAK) by western blot and the expression of phospho-tyrosine-FAK (pY397-FAK) in aldehyde dehydrogenase expressing (ALDH+) CSCs by flow cytometry. We created stable matched PDAC cell lines that overexpress full-length FAK (FAK-FL) or dominant-negative Y397F-FAK protein. We used these cell lines to study in vitro clonogenic growth and motility. We measured in vivo tumor-initiating cell frequency of these cell lines using a limiting dilution assay in NSG mice. We evaluated the efficacy of a small molecule inhibitor of pY397-FAK, PF573228, against PDAC CSCs in vitro.

Results: Among the panel of ECM proteins, type I collagen consistently increased in vitro clonogenic growth and motility of all three PDAC cell lines, with a concurrent increase in ALDH+ cells. The effects of type I collagen were specific to ALDH+ cells, since culturing ALDH+ cells on type I collagen led to higher retention of the CSC phenotype compared to culture under normal conditions, whereas culturing ALDHneg cells on type I collagen had no effect. Since ECM proteins can induce FAK activation via Y397-FAK autophosphorylation, we examined the expression of pY397-FAK in ALDH+ cells from cell lines and low-passage xenografts, and found that ALDH+ cells are 6-10-fold enriched in pY397-FAK expression compared to bulk PDAC cells (P&lt;.001). To investigate the role of FAK activity on CSC function, we overexpressed FAK-FL and Y397F-FAK in MIA-PaCa-2 and Capan-1 cells. Expression of pY397-FAK was 18-fold higher in FAK-FL-expressing cells but lost in Y397F-FAK-expressing cells. Compared to control cells, FAK-FL cells were 2-3 times (P&lt;0.001) more clonogenic and 1.5-2 times (P&lt;0.01) more motile, whereas Y397F-FAK cells were 1.6-5 times (P&lt;0.01) less clonogenic and 1.7-3 times (P&lt;0.01) less motile. FAK-FL expression enhanced the type I collagen-mediated increases in clonogenic growth and motility. In contrast, Y397F-FAK expression completely abrogated the effects of type I collagen. PF573228-treatment of PDAC cells inhibited clonogenic growth by 3-4-fold and motility by 3-fold and caused up to 40% loss of ALDH+ cells. Finally, we found that tumor-initiating cell frequency of MIAPaCa-2-control, MIAPaCa-2-FAK-FL, and MIAPaCa-2-pY397F-FAK cells was 1 in 350, 1 in 1, and 1 in 1150, respectively (P&lt;0.01).

Conclusion: Our studies show that interactions with the ECM enhance the number of PDAC CSCs, leading to increased clonogenic growth and cellular motility. We also show that FAK activation is necessary and sufficient for CSC function and may be a target of CSC-directed therapies.

Citation Format: Asma Begum, Theodore Ewachiw, Ross McMillan, Vesselin Penchev, Clinton Jung, Ally Huang, Jessica Norberg, NV Rajeshkumar, Anirban Maitra, William Matsui, Zeshaan A. Rasheed. The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A70.