Cancer-Stimulated Mesenchymal Stem Cells Create a Carcinoma Stem Cell Niche via Prostaglandin E

Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell–derived interleukin-1 (IL-1) induces prostaglandin E₂(PGE₂) secretion by MSCs. The resulting PGE₂ operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE₂ and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE₂ and cytokines. SIGNIFICANCE: Although PGE₂ has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE₂ axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells.Breast Cancer Research FoundationNational Institutes of Health (U.S.) (U54CA163109)Massachusetts Institute of Technology. Ludwig Center for Cancer Researc

ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways.

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia

Paradoxical decrease in norepinephrine content of adult mouse spleen and heart after neonatal nerve growth factor treatment

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21945/1/0000352.pd

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)- prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans

Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox.

Rationale: Endothelial cells (ECs) and platelets, which respectively produce antithrombotic prostacyclin and prothrombotic thromboxane A2, both express COX1 (cyclooxygenase1). Consequently, there has been no way to delineate any antithrombotic role for COX1-derived prostacyclin from the prothrombotic effects of platelet COX1. By contrast, an antithrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and antithrombotic protection in vivo. Objective: We sought to identify the role, if any, of COX1-derived prostacyclin in antithrombotic protection in vivo and compare this to the established protective role of COX2. Methods and Results: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic ECs released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in ECs but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, this effect was lost when COX1 was deleted from both ECs and platelets. Deletion of COX2 from ECs also resulted in a prothrombotic phenotype that was independent of local vascular prostacyclin production. Conclusions: These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential antithrombotic tone, which is masked when COX1 activity is lost in both ECs and platelets. These results help us define a new 2-component paradigm wherein thrombotic tone is regulated by both COX1 and COX2 through complementary but mechanistically distinct pathways

Profiling the eicosanoid networks that underlie the anti- and pro-thrombotic effects of aspirin

Aspirin prevents thrombosis by inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A2, a pro-thrombotic eicosanoid. However, the non-platelet actions of aspirin limit its antithrombotic effects. Here we used platelet-COX-1-ko mice to define the platelet and non-platelet eicosanoids affected by aspirin. Mass-spectrometry analysis demonstrated blood from platelet-COX-1-ko and global-COX- 1-ko mice produced similar eicosanoid profiles in vitro: e.g. formation of TxA2, prostaglandin (PG) F2, 11-HETE and 15-HETE was absent in both platelet- and global-COX-1-ko mice. Conversely, in vivo, platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-ko or aspirin-treated control mice, notably significantly higher levels of PGI2 metabolite. Ingenuity Pathway Analysis predicted that platelet-COX-1-ko mice would be protected from thrombosis, forming less prothrombotic TxA2 and PGE2. Conversely, aspirin or lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI2 and PGD2 at non-platelet sites leading to predicted thrombosis increase. In vitro and in vivo thrombosis studies proved these predictions. Overall, we have established the eicosanoid profiles linked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular system and linked them to anti- and pro-thrombotic effects of aspirin. These results explain why increasing aspirin dosage or aspirin addition to other drugs may lessen anti-thrombotic protection

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies

Development of New Deoxycytidine Kinase Inhibitors and Noninvasive in Vivo Evaluation Using Positron Emission Tomography

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC(50) = ∼1 – 12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy