Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

JQ1 and I-BET151 are selective inhibitors of BET bromodomain proteins that have efficacy against a number of different cancers. Since the effectiveness of targeted therapies is often limited by development of resistance, we examined whether it was possible for cancer cells to develop resistance to the BET inhibitor JQ1. Here we show that pancreatic cancer cells developing resistance to JQ1 demonstrate cross-resistance to I-BET151 and insensitivity to BRD4 downregulation. The resistant cells maintain expression of c-MYC, increase expression of JQ1-target genes FOSL1 and HMGA2, and demonstrate evidence of epithelial-mesenchymal transition (EMT). However, reverting EMT fails to sensitize the resistant cells to JQ1 treatment. Importantly, the JQ1-resistant cells remain dependent on c-MYC that now becomes co-regulated by high levels of GLI2. Furthermore, downregulating GLI2 re-sensitizes the resistant cells to JQ1. Overall, these results identify a mechanism by which cancer cells develop resistance to BET inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is associated with pronounced fibrosis that contributes to chemoresistance, in part, through increased histone acetylation. Since bromodomain (BRD) and extra terminal domain (BET) proteins are ‘readers’ of histone acetylation marks, we targeted BET proteins in PDAC cells grown in three-dimensional collagen. We show that treatment with BET inhibitors decreases growth of PDAC cells (AsPC1, CD18 and Panc1) in collagen. Transfection with siRNA against BRD4, which is increased in human PDAC tumors, also decreases growth of PDAC cells. BET inhibitors additionally decrease growth in collagen of PDAC cells that have undergone epithelial-to-mesenchymal transition or have become resistant to chemotherapy. Although BET inhibitors and BRD4 siRNA repress c-MYC only in AsPC1 and CD18 cells, downregulating c-MYC decreases growth of all three PDAC cell lines in collagen. FOSL1, which is also targeted by BET inhibitors and BRD4 siRNA in AsPC1, CD18 and Panc1 cells, additionally regulates growth of all three PDAC cell lines in collagen. BET inhibitors and BRD4 siRNA repress HMGA2, an architectural protein that modulates chromatin state and also contributes to chemoresistance, in PDAC cells grown in collagen. Importantly, we show that there is a statistically significant correlation between BRD4 and HMGA2 in human PDAC tumors. Significantly, overexpression of HMGA2 partially mitigates the effect of BET inhibitors on growth and c-MYC and/or FOSL1 expression in collagen. Overall, these results demonstrate that BET inhibitors block growth of PDAC cells in collagen and that BET proteins may be potential targets for the treatment of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. We have previously shown that PDAC cells are resistant to gemcitabine chemotherapy in the collagen microenvironment because of increased expression of the chromatin remodeling protein high mobility group A2 (HMGA2). We have now found that human PDAC tumors display higher levels of histone H3K9 and H3K27 acetylation in fibrotic regions. We show that relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels demonstrate increased histone H3K9 and H3K27 acetylation, along with increased expression of p300, PCAF and GCN5 histone acetyltransferases (HATs). Knocking down HMGA2 attenuates the effect of collagen on histone H3K9 and H3K27 acetylation and on collagen-induced p300, PCAF and GCN5 expression. We also show that human PDAC tumors with HMGA2 demonstrate increased histone H3K9 and H3K27 acetylation. Additionally, we show that cells in three-dimensional collagen gels demonstrate increased protection against gemcitabine. Significantly, down-regulation of HMGA2 or p300, PCAF and GCN5 HATs sensitizes the cells to gemcitabine in three-dimensional collagen. Overall, our results increase our understanding of how the collagen microenvironment contributes to chemo-resistance in vitro and identify HATs as potential therapeutic targets against this deadly cancer.

Patients with pancreatic cancer, which is characterized by an extensive collagen-rich fibrotic reaction, often present with metastases. A critical step in cancer metastasis is epithelial to mesenchymal transition, which can be orchestrated by Snail family of transcription factors. To understand the role of Snail (Snai1) in pancreatic cancer, we generated transgenic mice expressing Snail in the pancreas. While there was robust Snail expression, no phenotypic changes were observed. Since chronic pancreatitis can contribute to pancreatic cancer development, Snail-expressing mice were treated with cerulein to induce pancreatitis. Although there was significant tissue injury, no difference in pancreatitis was observed between control and Snail-expressing mice. As Kras mutation is necessary for tumor development in mouse models of pancreatic cancer, we generated mice expressing both mutant KrasG12D and Snail (Kras+/Snail+). Compared to control mice (Kras+/Snail−), Kras+/Snail+ mice developed acinar ectasia and more advanced acinar to ductal metaplasia. The Kras+/Snail+ mice exhibited increased fibrosis, increased pSmad2 levels and TGF-β2 expression, and activation of pancreatic stellate cells. To further understand how Snail promoted fibrosis, we established an in vitro model to examine the effect of Snail expression in pancreatic cancer cells on stellate cell collagen production. Snail expression in pancreatic cancer cells increased TGF-β2 levels and conditioned media from Snail-expressing pancreatic cancer cells increased collagen production by stellate cells. Additionally, inhibiting TGF-β signaling in stellate cells attenuated the conditioned media-induced collagen production by stellate cells. Together these results suggest that Snail contributes to pancreatic tumor development by promoting fibrotic reaction through increased TGF-β signaling.

ABSTRACTTheStaphylococcus aureusglobal regulator CodY responds to nutrient availability by controlling the expression of target genes.In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulencein vivo. The dominant community-associated methicillin-resistantS. aureus(CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that thecodYgenes of USA300 and other sequencedS. aureusisolates are not significantly different from thecodYgenes in strains known to have active CodY.codYwas expressed in USA300, as well as in other pulsotypes assessed. Deletion ofcodYfrom a USA300 clinical isolate resulted in modestly increased expression of the global regulatorsagrandsaeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of thelukF-PVgene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in thecodYmutant. All of these expression differences were reversed by complementation with a functionalcodYgene. Moreover, purified CodY protein bound upstream of thelukSF-PVoperon, indicating that CodY directly represses expression oflukSF-PV. Deletion ofcodYincreased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion oflukSF-PVfrom thecodYmutant did not attenuate virulence, indicating that the hypervirulence of thecodYmutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300.

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Human pancreatic ductal adenocarcinoma (PDAC) tumors are associated with dysregulation of mRNA translation. In this report, it is demonstrated that PDAC cells grown in collagen exhibit increased activation of the MAPK-interacting protein kinases (MNKs) that mediate eIF4E phosphorylation. Pharmacologic and genetic targeting of MNKs reverse epithelial-mesenchymal transition (EMT), decrease cell migration and reduce protein expression of the EMT-regulator ZEB1 without affecting ZEB1 mRNA levels. Paradoxically, targeting eIF4E – the best-characterized effector of MNKs – increases ZEB1 mRNA expression through repression of ZEB1-targeting microRNAs miR-200c and miR-141. In contrast, targeting the MNK effector hnRNPA1, which can function as a translational repressor, increases ZEB1 protein without increasing ZEB1 mRNA levels. Importantly, treatment with MNK inhibitors blocks growth of chemoresistant PDAC cells in collagen and decreases the number of aldehyde dehydrogenase activity-positive (Aldefluor+) cells. Significantly, MNK inhibitors increase E-cadherin mRNA levels and decrease vimentin mRNA levels in human PDAC organoids without affecting ZEB1 mRNA levels. Importantly, MNK inhibitors also decrease growth of human PDAC organoids. Implications These results demonstrate differential regulation of ZEB1 and EMT by MNKs and eIF4E, and identify MNKs as potential targets in pancreatic cancer.