BackgroundSoluble protein and lipid mediators play essential roles in the tumor environment, but their cellular origins, targets, and clinical relevance are only partially known. We have addressed this question for the most abundant cell types in human ovarian carcinoma ascites, namely tumor cells and tumor-associated macrophages.ResultsTranscriptome-derived datasets were adjusted for errors caused by contaminating cell types by an algorithm using expression data derived from pure cell types as references. These data were utilized to construct a network of autocrine and paracrine signaling pathways comprising 358 common and 58 patient-specific signaling mediators and their receptors. RNA sequencing based predictions were confirmed for several proteins and lipid mediators. Published expression microarray results for 1018 patients were used to establish clinical correlations for a number of components with distinct cellular origins and target cells. Clear associations with early relapse were found for STAT3-inducing cytokines, specific components of WNT and fibroblast growth factor signaling, ephrin and semaphorin axon guidance molecules, and TGFβ/BMP-triggered pathways. An association with early relapse was also observed for secretory macrophage-derived phospholipase PLA2G7, its product arachidonic acid (AA) and signaling pathways controlled by the AA metabolites PGE2, PGI2, and LTB4. By contrast, the genes encoding norrin and its receptor frizzled 4, both selectively expressed by cancer cells and previously not linked to tumor suppression, show a striking association with a favorable clinical course.ConclusionsWe have established a signaling network operating in the ovarian cancer microenvironment with previously unidentified pathways and have defined clinically relevant components within this network.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0956-6) contains supplementary material, which is available to authorized users.

Recently we identified GANT61, a small-molecule antagonist of Gli transcription factors, which are the final effectors of the mammalian Hedgehog (HH) signaling pathway. Here we describe a diamine substructure of GANT61 that carries the biological activity and show that this part of the molecule is structurally related to trans-1,4-bis(2-chlorobenzaminomethyl)cyclohexane dihydrochloride (AY9944), an inhibitor of the enzymatic activity and transcriptional inducer of 7-dehydrocholesterol-reductase (Dhcr7, EC 1.3.1.21). Treatment of cells with the GANT61 diamine, AY9944, or overexpression of DHCR7 results in the attenuation of Smoothened-dependent and -independent HH signaling. Whereas GANT61 function is independent of Dhcr7, AY9944 does require up-regulation of endogenous Dhcr7. In line with these findings, Dhcr7-modulating antipsychotic (clozapine, chlorpromazine, haloperidol) and antidepressant (imipramine) drugs regulate HH signaling in vitro and in vivo. Modulation of HH signaling may represent a hitherto undiscovered biological (side) effect of therapeutics used to treat schizophrenia and depression.

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.

DNA-damaging drugs induce a plethora of molecular and cellular alterations in tumor cells, but their interrelationship is largely obscure. Here, we show that carboplatin treatment of human ovarian carcinoma SKOV3 cells triggers an ordered sequence of events, which precedes the emergence of mitotic chemoresistant cells. The initial phase of cell death after initiation of carboplatin treatment is followed around day 14 by the emergence of a mixed cell population consisting of cycling, cell cycle-arrested and senescent cells. At this stage, giant cells make up >80% of the cell population, p21 (CDKN1A) in strongly induced, and cell numbers remain nearly static. Subsequently, cell death decreases, p21 expression drops to a low level and cell divisions increase, including regular mitoses of giant cells and depolyploidization by multi-daughter divisions. These events are accompanied by the upregulation of stemness markers and a pro-inflammatory secretory phenotype, peaking after approximately 14 days of treatment. At the same time the cells initiate epithelial to mesenchymal transition, which over the subsequent weeks continuously increases, concomitantly with the emergence of highly proliferative, migratory, dedifferentiated, pro-inflammatory and chemoresistant cells (SKOV3-R). These cells are anchorageindependent and grow in a 3D collagen matrix, while cells on day 14 do not survive under these conditions, indicating that SKOV3-R cells were generated thereafter by the multi-stage process described above. This process was essentially recapitulated with the ovarian carcinoma cell line IGROV-1. Our observations suggest that transitory cells characterized by polyploidy, features of stemness and a pro-inflammatory secretory phenotype contribute to the acquisition of chemoresistance.

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In a recent publication (1), we showed that low levels of the GLI1 transcription factor sensitize pancreatic ductal adenocarcinoma (PDAC) cells to induce an epithelial-to-mesenchymal transition (EMT). Analysis of the mechanism identified Ecadherin (encoded by the CDH1 gene) as a direct target of GLI1 in PDAC cells. We showed direct binding of GLI1 to the CDH1 promoter and transcriptional activation of the CDH1 gene by GLI1 in pancreatic cancer cells using expression and reporter studies. Finally, we were able to show a positive correlation between GLI1 and E-cadherin expression in cultured PDAC cells and in patient samples.Our data were questioned in a letter to the editor from Inaguma and colleagues who recently published that GLI1 induces mucin MUC5AC expression in PDAC cells leading to Ecadherin protein destabilization (2). In their letter, the authors raise concerns about several points of our manuscript, and we would like to reply to the major points of criticism in this response letter.One of these points refers to the fact that we did not show changes in E-cadherin levels upon GLI1 overexpression in several PDAC cell lines, a somewhat astonishing criticism given the fact that the authors themselves never properly showed alterations in E-cadherin expression upon full-length GLI1 overexpression in their own manuscript (2). However, we assume that the transcriptional machinery that controls the GLI1-CDH1 axis might be saturated in these cells and therefore no change in CDH1 levels upon further increase of GLI1 by means of transfection can be seen. One exception was MiaPaca-2 cells, which upregulated CDH1 expression upon GLI1 transfection, despite the reported hypermethylation of the CDH1 promoter in these cells. In agreement with our data, others have also reported induction of CDH1 in MiaPaca-2 cells upon transfection with transcription factor expression plasmids, suggesting that the promoter methylation does not fully prevent transcriptional activity at this gene locus under certain circumstances (3). Also not discussed in the letter by Inaguma and colleagues is our finding that a transcriptionally inactive GLI1 lacking the DNA-binding domain is able to suppress E-cadherin expression and induce an EMT in Panc1 cells. We interpret these data as dominant-negative behavior of the mutant GLI1 on the wild-type form. Taken together, we concluded that in most of the cell lines analyzed, the effects of GLI1 on CDH1 are saturated, and only a reduction in GLI1 is translated into effects on CDH1. A slightly alternative possible scenario would be that GLI1 is required to maintain CDH1 expression once the promoter has been activated by a second factor. In such a case, the second factor would define the level of CDH1 expression and a reduction in the maintenance factor (GLI1) would decrease the CDH1 expression but increasing GLI1 levels would not further stimulate it.Another point of concern that Inaguma and colleagues pointed out was that our clinical samples were not microdissected and therefore the positive correlati...

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