Primary PDA cells in culture do not express Rgs16::GFP when grown in media with 5% FBS (Fig.?2G), but Rgs16::GFP expression is induced PKC-IN-1 within 16?h of treatment with 50% FBS (Fig.?2H). vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem?+?TSA?+?JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in PKC-IN-1 vivo screen. mice (p48Cre/+the combination of GEM?+?TSA?+?JQ1 significantly reduced initiation and growth of spontaneous tumors. Here we demonstrate an effective screen for novel PDA therapeutics. First, primary PDA cells in culture are screened for small molecules that induce Rgs16::GFP expression in response to stress. Molecules that synergize with Gem, a standard-of-care cytotoxic drug are identified in cell culture viability assays. Efficacy in mice is validated in a rapid in vivo?assay of PDA initiation and growth. These steps provide a quick and efficient approach for identifying new and effective therapeutic combinations to treat PDA. Results Alterations in HDAC activity occur in numerous cancers and have prompted the search for pharmacological agents capable of inhibiting these enzymes24,25. Several studies have reported elevated expression of HDACs and BETs in PDA. HDAC1, 2, 3, 4, and 7 were reported to be upregulated in PDA, whereas HDAC 2 and 3, along with SIRT1, have been reported to be involved in cancer invasion and chemo-resistance31C35. Thus, we assessed the differential expression profile of all HDACs and BETs in human PDA tissue samples in the TCGA database and compared these to mouse models of caerulein-treated pancreatitis, PDA (KIC), and primary PDA cells from KIC mice. HDACs and BET proteins are highly expressed in human and mouse PDA We analyzed the differential expression of HDACs and BETs at various stages of disease progression in mice. First, we compared expression in normal untreated (UT) pancreas of adult mice to pancreata collected from mice injected (i.p.) with caerulein 2, 4, and 7?days post-treatment (Fig.?1A). Acinar-to-ductal metaplasia (ADM) is greatest at d2 post-caerulein, and morphology gradually returns to normal as the exocrine pancreas recovers by PKC-IN-1 day 736. Expression of nearly all HCAC and BET genes reflects this pattern, showing highest expression at day 2, sequentially declining towards normal levels at days 4 and 7. Open in a separate window Figure 1 HDAC and BET family of bromodomain protein expression in caerulein induced acute pancreatitis, human PDA, and mouse PDA. Relative expression of HDACs and BET family bromodomain proteins were analyzed in (A) wild-type untreated (UT) pancreas and 2, 4, 7?days post caerulein injections, (B) mouse primacy PDA cells and, (C) human PDA (72 samples in TCGA database).?Primary PDA cells from?mice (E) early lesions and (F) late tumors. HDAC and BET bromodomain protein genes are differentially expressed in the identified cell populations analyzed by scRNAseq. Each column represents an individual cell, and each row is the expression value for a single gene. Genes are listed in the same order in (ACE). Violin plots show a representative HDAC (HDAC1) and BET family bromodomain protein 2 (Brd2) from each sample. Cell types are (D) A, acinar; I, islet endocrine cells; F1-F3, fibroblasts; M, macrophage; TC, T cells; BC, B cells. (E) I, islet endocrine other than beta cells; IB; endocrine beta cells; EC; epithelial cancer; E, endothelial cells. (F) MC, mesenchymal cancer cells; L, Lymphocytes (Treg). Analysis and figure generation were performed using R MGC45931 statistical software [R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/]. HDAC and BET expression in early stage ADM was compared to primary PDA cells from KIC mice (Fig.?1B) and human tumor samples (Fig.?1C) in the TCGA dataset (n?=?72) collected predominately from stage 1 and 2 patients. In mice, the relative expression of HDAC and BET proteins is similar, except for the reciprocal pattern of HDAC1 and HDAC11 in ADM and primary PDA cancers (Fig.?1B). Primary cancer cells isolated from KIC-Rgs16::GFP mice were sorted for high and low expression of Rgs16::GFP (2.6-fold differential expression following 12?h incubation in 50% or 5% FBS, respectively) (Fig.?1B). By contrast,.
Primary PDA cells in culture do not express Rgs16::GFP when grown in media with 5% FBS (Fig
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