Abstract A70: The extracellular matrix regulates pancreatic cancer stem cell function via focal adhesion kinase signaling.

Abstract

Introduction: Pancreatic adenocarcinoma is a highly aggressive disease with a propensity for metastasis and drug resistance early in its course. Pancreatic cancer stem cells (CSCs) may play a role in these processes. We previously found that pancreatic CSCs can be isolated based on aldehyde dehydrogenase (ALDH) expression and that these cells are relatively more invasive and drug resistant compared to their ALDHneg counterparts. Though generally uncommon (usually <5% of all cells), ALDH+ cells are more frequently located at the invasive edge of tumors. Normal stem cell function is primarily regulated by the extracellular stem cell niche, and the densely fibrotic desmoplastic reaction (DR), composed of non-malignant cells and specific extracellular matrix (ECM) proteins, is a hallmark of pancreatic tumors; therefore, we examined the role of the ECM on pancreatic CSC maintenance and function. Methods: We utilized several cell lines and patient xenografts for our studies. To determine the effect of different ECM proteins on pancreatic cancer cells, we cultured cells on ECM-coated plates (BD Biosciences) and harvested them for further analyses. Colony formation and long-term self-renewal was measured by culturing cells in methylcellulose with serial re-plating. In vitro migration was measured by counting the number of cells able to traverse transwell filters containing 8-μm pores (BD Biosciences). We immunophenotyped CSCs using the ALDEFLUOR reagent, antibodies against α2β1 integrin (Santa Cruz Biotechnology) and phospho-397-focal adhesion kinase (FAK; Cell Signaling Technology), and a FACSCalibur flow cytometer (BD Biosciences). We studied the loss of FAK signaling in pancreatic cancer cells by using a specific small molecule inhibitor, PF573228 (Tocris Bioscience), and several pancreatic cancer cell lines that we made to constitutively overexpress a fragment of FAK lacking the tyrosine kinase domain, which functions as a dominant negative protein. Results: Compared to cells cultured under standard conditions, only type I collagen resulted in cells having an increased proportion of ALDH+ cells with concomitant increases in clonogenic growth and migratory potential. To determine if collagen provides a selective advantage to ALDH+ cells or induced ALDHneg cells to acquire a CSC phenotype, we isolated ALDH+ and ALDHneg cells and cultured them on plates coated with type I collagen. We found that ALDH+ cells maintained their CSC phenotype to a higher degree (2-3 fold) in the presence of type I collagen compared to those cultured on plastic or Matrigel. In contrast, ALDHneg cells cultured on collagen remained phenotypically similar to those cultured on plastic or Matrigel. Since type I collagen has been shown to mediate its effects on cells via α2β1 integrin and FAK, we examined the expression of these proteins in ALDH+ and ALDHneg cells. We found that expression of the α2β1 integrin heterodimer was restricted to ALDH+ cells and that ALDH+ cells expressed higher levels of activated, phosphorylated FAK compared to the bulk tumor cell population. Pharmacologic inhibition of FAK activation resulted in the loss of ALDH+ cells, clonogenic growth, and cellular migration. Likewise, a dominant-negative form of FAK, lacking the tyrosine kinase domain, resulted in similar functional deficits. Conclusions: Our data demonstrates that type I collagen, a key component of the pancreatic cancer DR, supports the maintenance of ALDH+ pancreatic CSCs that results in enhanced tumorigenic and metastatic potential of these cells. Furthermore, disruption of FAK signaling can inhibit pancreatic CSC function and may serve as a novel CSC-directed therapeutic strategy. Citation Format: Zeshaan A. Rasheed, Clinton Jung, Ally Huang, Jessica Norberg, N.V. Rajeshkumar, Anirban Maitra, William Matsui. The extracellular matrix regulates pancreatic cancer stem cell function via focal adhesion kinase signaling. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A70.