: CXCL10 or the Interferon gamma-inducible Protein 10 (IP10) belongs to the CXC subfamily of chemokines. Classically, chemokines are known to modulate leukocyte trafficking of cells expressing corresponding receptors. CXCL10 expression in pancreatic ductal adenocarcinoma (PDA) patient samples has been correlated with CXCR3 (receptor for CXCL10) positive regulatory T cell (Treg) infiltration contributing to an immuno-suppressed tumor microenvironment. However, other studies suggest a more direct and non-classical role of CXCL10 in promoting acinar cell injury and apoptosis. Chronic pancreatitis patients have also been shown to express high levels of CXCL10 which is suggestive of its role in the pathogenesis of the disease. Clinically, there is a significant overlap between chronic pancreatitis and pancreatic cancer, but the role of CXCL10 specifically in the context of genetic mutations causing pancreatic cancer has not been fully explored. Our preliminary in situ hybridization data show that the cells in PanIN lesions of p48Cre;LSL-KrasG12D mice express CXCL10 in contrast to no expression seen in normal adjacent acinar cells, whereas the receptor CXCR3 is highly expressed in the normal adjacent acinar cells as compared to that in the precursor lesion cells. Induction of CXCL10 expression in the PanIN cells and expression of CXCR3 in normal adjacent acinar cells suggests an important role of this chemokine in exacerbating the inflammatory pancreatic environment during the development of pancreatic cancer. This study aims to (I) delineate the mechanism of induction of CXCL10 expression in areas with precursor lesion; (II) determine the effect of CXCL10 on normal acinar cells; and (III) test if CXCL10 neutralization in the pancreas has an effect on the progression of the disease.
Epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis.Previous studies have shown that MCF10A human mammary epithelial cells undergo EMT when cultured at low cell density, but the underlying mechanism remains poorly understood.Here we show that the expression of proteasome genes and the proteasome activity increase in response to low cell density.Moreover, the proteasome inhibitor MG132 blocks EMT in sparse MCF10A cells.The transcription of E-cadherin gene is repressed in sparse MCF10A cells, but is recovered upon the treatment with MG132.While the mRNA levels of Snail and Slug are markedly elevated at low cell density, the steady state protein levels are similar between sparse and confluent cells.These results suggest that the suppression of E-cadherin gene expression in sparse MCF10A cells is likely caused by downregulation of its transcription activator(s) rather than upregulation of its repressors such as Snail and Slug.This study reveals that cell density-dependent EMT in MCF10A cells is regulated by proteasome activity.
The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.
Oncogenic mutations of KRAS are the most frequent driver mutations in pancreatic cancer. Expression of an oncogenic allele of KRAS leads to metabolic changes and altered cellular signaling that both can increase the production of intracellular reactive oxygen species (ROS). Increases in ROS have been shown to drive the formation and progression of pancreatic precancerous lesions by upregulating survival and growth factor signaling. A key issue for precancerous and cancer cells is to keep ROS at levels where they are beneficial for tumor development and progression, but below the threshold that leads to induction of senescence or cell death. In KRas-driven neoplasia aberrantly increased ROS levels are therefore balanced by an upregulation of antioxidant genes.
Background: CD36 is a scavenger and antiangiogenic receptor that plays an important role in athero-thrombotic diseases, diabetes and cancer and contributes to obesity. Lysophosphatidic acid (LPA), a bioactive phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by down-regulating endothelial CD36 transcription via protein kinase PKD-1 signaling. However, the precise mechanism as to how angiogenic signaling is integrated to regulate endothelial specific CD36 transcription remain unknown. Hypothesis: LPA represses CD36 transcription through PKD-1-mediated formation of a nuclear transcriptional complex in endothelial cells. Methods: Microvascular endothelial cells expressing CD36 were used for studying signaling and CD36 transcription by real time RT-qPCR, Western blotting, co-immunoprecipitation or avidin-biotin-conjugated DNA-binding assay; angiogenesis gene array was used for angiogenic gene profiling in response to LPA exposure. Spheroid-based angiogenesis assay, in vivo Matrigel assay and tumor angiogenesis model in CD36 deficiency and wild type mice were established to elucidate mechanisms of angiogenic signaling. Results: CD36 transcriptional repression involved PKD-1 signaling mediated formation of FoxO1-HDAC7 complex in the nucleus of endothelial cells. Unexpectedly, turning off CD36 transcription initiated reprogramming MVECs to express ephrin B2, a critical “molecular signature” involved in angiogenesis and arteriogenesis, and increased phosphorylation of Erk1/2, the MAP kinase important in arterial differentiation. PKD-1 signaling was also shown in tumor endothelium of Lewis lung carcinomas, along with low CD36 expression or CD36 deficiency. Angiogenic branching morphogenesis and in vivo angiogenesis were dependent on PKD-1 signaling. Conclusion: LPA/PKD1-HDAC7-FoxO1 signaling axis regulates endothelial CD36 transcription and mediates silencing of the antiangiogenic switch, resulting in proarteriogenic reprogramming. Targeting this signaling cascade could be a novel approach for cancer, diabetes, athero-thrombotic diseases and obesity.
Efficient elimination of mitochondrial reactive oxygen species (mROS) correlates with increased cellular survival and organism life span. Detoxification of mitochondrial ROS is regulated by induction of the nuclear SOD2 gene, which encodes the manganese-dependent superoxide dismutase (MnSOD). However, the mechanisms by which mitochondrial oxidative stress activates cellular signaling pathways leading to induction of nuclear genes are not known. Here we demonstrate that release of mROS activates a signal relay pathway in which the serine/threonine protein kinase D (PKD) activates the NF-kappaB transcription factor, leading to induction of SOD2. Conversely, the FOXO3a transcription factor is dispensable for mROS-induced SOD2 induction. PKD-mediated MnSOD expression promotes increased survival of cells upon release of mROS, suggesting that mitochondrion-to-nucleus signaling is necessary for efficient detoxification mechanisms and cellular viability.
In response to inflammation, pancreatic acinar cells can undergo acinar-to-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a ductlike phenotype and, in the context of additional oncogenic stimulation, contributes to development of pancreatic cancer. The signaling mechanisms underlying pancreatitis-inducing ADM are largely undefined. Our results provide evidence that macrophages infiltrating the pancreas drive this transdifferentiation process. We identify the macrophage-secreted inflammatory cytokines RANTES and tumor necrosis factor α (TNF) as mediators of such signaling. Both RANTES and TNF induce ADM through activation of nuclear factor κB and its target genes involved in regulating survival, proliferation, and degradation of extracellular matrix. In particular, we identify matrix metalloproteinases (MMPs) as targets that drive ADM and provide in vivo data suggesting that MMP inhibitors may be efficiently applied to block pancreatitis-induced ADM in therapy.
