Abstract Nodal and activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and/or Activin regulate self-renewal and invasiveness of pancreatic cancer stem cells. While their expression was hardly detectable in differentiated pancreatic cancer cells, cancer stem cells demonstrate a drastic upregulation for Nodal and, to a lesser extent, Activin while TGF-β remained unchanged. Pancreatic stellate cells as the putative microenvironment for cancer stem cells produce very high amounts of Activin, and, to a lesser extent, Nodal. Knockdown and pharmacological inhibition of their common receptor Alk4/7 both significantly, but reversibly reduced cancer stem cell self-renewal / invasiveness and cancer stem cells subsequently became sensitive to gemcitabine. Importantly, while orthotopically engrafted primary human cancer cell suspensions were highly responsive to Nodal/Activin inhibition plus gemcitabine, engrafted primary human pancreatic cancer tissue xenografts containing massive stroma did not slow down tumor progression. Intriguingly, the addition of a stroma-targeting sonic hedgehog pathway inhibitor resulted in enhanced delivery of the Nodal/Activin inhibitor and gemcitabine, respectively, and translated into long-term disease stabilization. Therefore, modulation of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a novel therapeutic strategy for targeted elimination of cancer stem cells in order to overcome their resistance towards gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B45.

Nodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.

The identification of novel approaches to specifically target the DNA-damage checkpoint response in chemotherapy-resistant cancer stem cells (CSC) of solid tumors has recently attracted great interest. We show here in colon cancer cell lines and primary colon cancer cells that inhibition of checkpoint-modulating phosphoinositide 3-kinase-related (PIK) kinases preferentially depletes the chemoresistant and exclusively tumorigenic CD133(+) cell fraction. We observed a time- and dose-dependent disproportionally pronounced loss of CD133(+) cells and the consecutive lack of in vitro and in vivo tumorigenicity of the remaining cells. Depletion of CD133(+) cells was initiated through apoptosis of cycling CD133(+) cells and further substantiated through subsequent recruitment of quiescent CD133(+) cells into the cell cycle followed by their elimination. Models using specific PIK kinase inhibitors, somatic cell gene targeting, and RNA interference demonstrated that the observed detrimental effects of caffeine on CSC were attributable specifically to the inhibition of the PIK kinase ataxia telangiectasia- and Rad3-related (ATR). Mechanistically, phosphorylation of CHK1 checkpoint homolog (S. pombe; CHK1) was significantly enhanced in CD133(+) as compared with CD133(-) cells on treatment with DNA interstrand-crosslinking (ICL) agents, indicating a preferential activation of the ATR/CHK1-dependent DNA-damage response in tumorigenic CD133(+) cells. Consistently, the chemoresistance of CD133(+) cells toward DNA ICL agents was overcome through inhibition of ATR/CHK1-signaling. In conclusion, our study illustrates a novel target to eliminate the tumorigenic CD133(+) cell population in colon cancer and provides another rationale for the development of specific ATR-inhibitors.

Over the past decade, increasing evidence suggested that stem cells play a crucial role not only in the generation of complex multicellular organisms, but also in the development and progression of malignant diseases. For the most abundant tumours, it has been shown that they contain a subset of distinct cancer cells that is exclusively responsible for tumour initiation and propagation These cells are termed cancer stem cells or tumour-initiating cells and they are also highly resistant to chemotherapeutic agents. Because CSC are preferentially endowed with the self-renewal capacity, it has further been hypothesized that they are also exclusively responsible for metastasis. Indeed, we were able to show that pancreatic cancer stem cells contain a subpopulation of migrating cancer stem cells characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumour and metastasizing. Laboratories around the world are now aiming to further characterize these cells to eventually identify novel treatment modalities to fight cancer. Thus, cancer stem cells are promising new targets to counteract the growth-promoting and metastatic potential of solid tumours.

BACKGROUND & AIMSPancreatic cancers contain exclusively tumorigenic cancer stem cells (CSCs), which are highly resistant to chemotherapy, resulting in a relative increase in CSC numbers during gemcitabine treatment. Signaling through sonic hedgehog and mammalian target of rapamycin (mTOR), respectively, may be essential for CSC self-renewal and could represent putative targets for novel treatment modalities.METHODSWe used in vitro and in vivo models of pancreatic cancer to examine the effects of sonic hedgehog inhibition (cyclopamine/CUR199691) and mTOR blockade (rapamycin) on the tumorigenic CSC population.RESULTSSurprisingly, neither cyclopamine nor rapamycin alone or as supplements to chemotherapy were capable of effectively diminishing the CSC pool. Only the combined inhibition of both pathways together with chemotherapy reduced the number of CSCs to virtually undetectable levels in vitro and in vivo. Most importantly, in vivo administration of this triple combination in mice with established patient-derived pancreatic tumors was reasonably tolerated and translated into significantly prolonged long-term survival.CONCLUSIONSThe combined blockade of sonic hedgehog and mTOR signaling together with standard chemotherapy is capable of eliminating pancreatic CSCs. Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the devastating prognosis of patients with pancreatic cancer.

Background: Solid tumours are the most common cancers and represent a major therapeutic challenge. The cancer stem cell (CSC) hypothesis is an attractive model to explain the functional heterogeneity commonly observed in solid tumours. It proposes a hierarchical organization of tumours, in which a subpopulation of stem cell-like cells sustains tumour growth, metastasis and resistance to therapy. Objective: Here we review the most recent advances in the CSC field, with particular emphasis on pancreatic cancer as one of the deadliest human tumours, and highlight open questions and caveats to be addressed in future studies. Methods: This review focuses on the role of CSC in the promotion and metastasis of solid tumours and summarizes recent findings regarding the targeting of signalling pathways that are of particular importance for the maintenance and the elimination of CSC as the proposed root of the tumour. Results/conclusions: There is increasing evidence that solid tumours, including pancreatic cancer, are hierarchically organized and sustained by a distinct subpopulation of CSC. Direct evidence for the CSC hypothesis has emerged from mouse models only recently. While the clinical relevance of CSC remains a fundamental issue, current findings suggest that specific targeting of these cells is possible and therapeutically relevant.