Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). METHODS: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against ß1 integrin and FAK as well as a small-molecule FAK inhibitor. RESULTS: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of ß1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. CONCLUSION: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

Ezrin Promotes Stem Cell Properties in Pancreatic Ductal Adenocarcinoma.

Self-renewal maintains the long-term clonogenic growth that is required for cancer relapse and progression, but the cellular processes regulating this property are not fully understood. In many diseases, self-renewal is enhanced in cancer stem cells (CSC), and in pancreatic ductal adenocarcinoma (PDAC), CSCs are characterized by the surface expression of CD44. In addition to cell adhesion, CD44 impacts cell shape and morphology by modulating the actin cytoskeleton via Ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family of linker proteins. We examined the expression of Ezrin in PDAC cells and found higher levels of both total and activated Ezrin in CSCs compared with bulk tumor cells. We also found that the knockdown of Ezrin in PDAC cells decreased clonogenic growth, self-renewal, cell migration, and CSC frequency in vitro as well as tumor initiation in vivo. These effects were associated with cytoskeletal changes that are similar to those occurring during the differentiation of normal stem cells, and the inhibition of actin remodeling reversed the impact of Ezrin loss. Finally, targeting Ezrin using a small-molecule inhibitor limited the self-renewal of clinically derived low-passage PDAC xenografts. Our findings demonstrate that Ezrin modulates CSCs properties and may represent a novel target for the treatment of PDAC. IMPLICATIONS: Our findings demonstrate that Ezrin modulates CSCs' properties and may represent a novel target for the treatment of PDAC.

IQGAP1 Scaffold-MAP Kinase Interactions Enhance Multiple Myeloma Clonogenic Growth and Self-Renewal.

Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Mol Cancer Ther; 15(11); 2733-9. ©2016 AACR.

Heterogeneity and targeting of pancreatic cancer stem cells.

Cancer stem cells (CSC) have been identified in an ever-increasing number of human malignancies on the basis of their ability to recapitulate tumors in the ectopic setting and maintain long-term tumorigenic potential. In addition, in pancreatic adenocarcinoma, CSCs may display additional properties, such as relative drug resistance and enhanced invasive and migratory potential that implicate a role in disease pathogenesis spanning initial tumor formation to metastatic disease progression. Importantly, these findings also indicate that the development of novel therapeutic strategies capable of inhibiting or eliminating CSCs will improve clinical outcomes. Preclinical studies have already described a wide array of potential approaches that target CSC-specific surface antigens and cellular pathways involved in cell survival, adhesion, self-renewal, and differentiation. Further, progress in this area should continue to move forward as the unique biology of CSCs is better understood. All preclinical studies to date have focused on targeting specific and phenotypically defined CSCs, but multiple cell populations with the ability to form tumors and self-renew have been identified in pancreatic carcinoma. As the clinical efficacy of CSC-directed therapies will depend on the inhibition of all sources of tumor self-renewal, better understanding of how specific CSC populations are related to one another and whether each possesses specific functional properties will be critical. In this CCR Focus article, we discuss the potential relationships between different pancreatic CSC populations and strategies to identify novel targeting approaches.

Human embryonic stem cell-derived CD34+ cells function as MSC progenitor cells.

Mesenchymal stem/stromal cells (MSCs) have been isolated from various tissues and utilized for an expanding number of therapies. The developmental pathways involved in producing MSCs and the phenotypic precursor/progenitor cells that give rise to human MSCs remain poorly defined. Human embryonic stem cells (hESCs) have the capability to generate functional hemato-endothelial cells and other mesoderm lineage cells. hESC-derived CD73(+) cells have been isolated and found to have similar phenotypic and functional characteristics as adult MSCs. Here we demonstrate hESC-derived CD34(+)CD73(-) cells can serve as MSC progenitor cells with the ability to differentiate into adipocytes, osteoblasts and chondrocytes. Additionally, gene array analysis of hESC-derived MSCs show substantially different gene expression compared to bone marrow (BM)-derived MSCs, especially with increased expression of pluripotent and multipotent stem cell and endothelial cell-associated genes. The isolation of functional MSCs from hESC-derived CD34(+)CD73(-) cells provides improved understanding of MSC development and utilization of pluripotent stem cells to produce MSCs suited for novel regenerative therapies.

Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34(+) cells preferentially develop into endothelial cells.

Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow noninvasive, real-time bioluminescent imaging of hESC-derived CD34(+) cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc(+) hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34(+) cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. Although transplanted hESC-derived CD34(+) cells are well-suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment.