Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.

ObjectiveCancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes.DesignMicro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs.ResultsWe found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-β1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer.ConclusionsOur findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.

Esta es la versión de autor del artículo publicado en: This is an author produced version of a paper published in: Gut 64.12 (2015Gut 64.12 ( ): 1921Gut 64.12 ( -1935 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Results:We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to TGF-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSC with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion, and tumourigenicity via formyl peptide receptor 2 (FPR2)-and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from CRAMP (i.e murine homolog of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. Conclusion:Thus, hCAP-18/LL-37 represents a previously unrecognized PDAC micro-environment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis. SIGNIFICANCE OF THE STUDYWhat is already known on this subject?• Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with limited therapeutic options.• Pancreatic cancer stem cells (CSCs) are exclusively tumourigenic and highly resistant to chemotherapy.• Tumour-associated macrophages are important for the progression and metastatic spread of many solid tumours. What are the new findings?• The immuno-modulatory cationic antimicrobial peptide 18/leucine leucine-37 (hCAP-18/LL-37) is over expressed in the stroma of PDAC and acts on CSCs to potentiate their inherent biological properties.• Tumour-associated macrophages secrete hCAP-18/LL-37 in direct response to CSC-secreted NODAL/ACTIVINA/TGF-β1.• Small molecule targeting of the LL-37 receptors formyl peptide receptor 2 (FPR2) and P2X purinoceptor 7 receptor (P2X7R), present on pancreatic CSCs, negatively impacts tumour growth and circulating tumour cell numbers. How might it impact on clinical practice in the foreseeable future?• The discovery of the crucial role of hCAP-18/LL-37 in cancer stem cell biology represents an important advancement in our understanding of the PDAC tumour microenvironment.• Targeting pancreatic CSCs using inhibitors of the LL-37 receptors FPR2 and P2X7R may represent a specific therapeutic approach to block the tumour promoting cross-talk that exists within the tumour microenviro...

Phosphorylation of histone H2AX (cH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of cH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal cH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining selfrenewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of cH2AX. We show that basal cH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with selfrenewal-enhancing small molecules. We observe that selfrenewal activity is highly compromised in H2AX2/2 cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.