A miRNA signature of chemoresistant mesenchymal phenotype identifies novel molecular targets associated with advanced pancreatic cancer.

In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.

Inhibiting signal transducer and activator of transcription-3 increases response to gemcitabine and delays progression of pancreatic cancer.

BACKGROUND: Among the solid tumors, human pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis. Gemcitabine is the standard first line of therapy for pancreatic cancer but has limited efficacy due to inherent or rapid development of resistance and combining EGFR inhibitors with this regimen results in only a modest clinical benefit. The goal of this study was to identify molecular targets that are activated during gemcitabine therapy alone or in combination with an EGFR inhibitor. METHODS: PDAC cell lines were used to determine molecular changes and rates of growth after treatment with gemcitabine or an EGFR inhibitor, AG1478, by Western blot analysis and MTT assays respectively. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after gemcitabine treatment. ShRNA was used to knockdown STAT3. An in vivo orthotopic animal model was used to evaluate STAT3 as a target. Immunohistochemical analysis was performed to analyze Ki67 and STAT3 expression in tumors. RESULTS: Treatment with gemcitabine increased the levels of EGFRTyr1068 and ERK phosphorylation in the PDAC cell lines tested. The constitutive STAT3Tyr705 phosphorylation observed in PDAC cell lines was not altered by treatment with gemcitabine. Treatment of cells with gemcitabine or AG1478 resulted in differential rate of growth inhibition. AG1478 efficiently blocked the phosphorylation of EGFRTyr1068 and inhibited the phosphorylation of down-stream effectors AKT and ERKs, while STAT3Tyr705 phosphorylation remained unchanged. Combining these two agents neither induced synergistic growth suppression nor inhibited STAT3Tyr705 phosphorylation, thus prompting further studies to assess whether targeting STAT3 improves the response to gemcitabine or AG1478. Indeed, knockdown of STAT3 increased sensitivity to gemcitabine by inducing pro-apoptotic signals and by increasing G1 cell cycle arrest. However, knockdown of STAT3 did not enhance the growth inhibitory potential of AG1478. In vivo orthotopic animal model results show that knockdown of STAT3 caused a significant reduction in tumor burden and delayed tumor progression with increased response to gemcitabine associated with a decrease in the Ki-67 positive cells. CONCLUSIONS: This study suggests that STAT3 should be considered an important molecular target for therapy of PDAC for enhancing the response to gemcitabine.

Inhibition of STAT3 Tyr705 phosphorylation by Smad4 suppresses transforming growth factor beta-mediated invasion and metastasis in pancreatic cancer cells.

The role of Smad4 in transforming growth factor beta (TGFbeta)-mediated epithelial-mesenchymal transition (EMT), invasion, and metastasis was investigated using isogenically matched pancreatic cancer cell lines that differed only in expression of Smad4. Cells expressing Smad4 showed an enhanced TGFbeta-mediated EMT as determined by increased expression of vimentin and decreased expression of beta-catenin and E-cadherin. TGFbeta-mediated invasion was suppressed in Smad4-intact cells as determined by in vitro assays, and these cells showed a reduced metastasis in an orthotopic model of pancreatic cancer. Interestingly, TGFbeta inhibited STAT3(Tyr705) phosphorylation in Smad4-intact cells. The decrease in STAT3(Tyr705) phosphorylation was linked to a TGFbeta/Smad4-dependent and enhanced activation of extracellular signal-regulated kinases, which caused an increase in serine phosphorylation of STAT3(Ser727). Down-regulating signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA in Smad4-deficient cells prevented TGFbeta-induced invasion. Conversely, expressing a constitutively activated form of STAT3 (STAT3-C) in Smad4-intact cells enhanced invasion. This study indicates the requirement of STAT3 activity for TGFbeta-induced invasion in pancreatic cancer cells and implicates Smad4-dependent signaling in regulating STAT3 activity. These findings further suggest that loss of Smad4, leading to aberrant activation of STAT3, contributes to the switch of TGFbeta from a tumor-suppressive to a tumor-promoting pathway in pancreatic cancer.

Downregulation of PAR-4, a pro-apoptotic gene, in pancreatic tumors harboring K-ras mutation.

Oncogenic ras is known to inhibit cell death and growth inhibitory genes and activate prosurvival genes. Proapoptotic gene PAR-4, has been found to be downregulated by oncogenic ras. Since pancreatic tumors harbor a high incidence of K-ras point mutations, we hypothesized that oncogenic K-ras might influence the function and expression of PAR-4. PAR-4 expression levels were analyzed in 4 established pancreatic tumor cell lines, 10 normal pancreatic tissues, 44 frozen tumor tissues and 25 paraffin-embedded pancreatic adenocarcinoma samples by Real Time RT-PCR, Western blot analysis and immunohistochemistry. K-ras mutational status was analyzed by allele-specific oligonucleotide-hybridization. Expression levels of PAR-4 were correlated with the K-ras mutational status and clinical characteristics. Further, modulation of endogenous PAR-4 was tested by transiently expressing oncogenic ras in a wild-type K-ras pancreatic cancer cell line, BxPC-3. Three cell lines with K-ras mutations showed low levels of PAR-4 when compared to a normal pancreatic tissue. Of 44 frozen tumors, 16 showed appreciable upregulation of Par mRNA and 27 showed significant downregulation of PAR-4 mRNA when compared to normal pancreatic tissue and 1 had levels equivalent to normal pancreatic tissue. Of 25 paraffin-embedded tumors, 9 showed downregulation of PAR-4 protein and this downregulation of PAR-4 correlated significantly with K-ras mutational status (p < 0.00002). In addition, the presence of PAR-4 mRNA or protein expression in pancreatic tumors correlated with prolonged survival. Transient overexpression of oncogenic ras in wild-type K-ras BxPC-3 cells significantly downregulated the endogenous PAR-4 protein levels and conferred accelerated growth. Thus, downregulation or loss of PAR-4 expression by oncogenic ras may provide a selective survival advantage for pancreatic tumors, through inhibition of proapoptotic pathway mediated by PAR-4.

Farnesyl transferase inhibitor (R115777)-induced inhibition of STAT3(Tyr705) phosphorylation in human pancreatic cancer cell lines require extracellular signal-regulated kinases.

In this study, we report that R115777, a nonpeptidomimetic farnesyl transferase inhibitor, suppresses the growth of human pancreatic adenocarcinoma cell lines and that this growth inhibition is associated with modulation in the phosphorylation levels of signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinases (ERK). Treatment of cells with R115777 inhibited the tyrosine phosphorylation of STAT3((Tyr705)), while increasing the serine phosphorylation of STAT3((Ser727)). We found the differential phosphorylation of STAT3 was due to an increased and prolonged activation of ERKs. The biological significance of ERK-mediated inhibition of STAT3((Tyr705)) phosphorylation was further assessed by treating the cells with an inhibitor (PD98059) of mitogen-activated protein kinase kinase (MEK) or by transfecting the cells with a vector that expresses constitutively active MEK-1. Expression of constitutively active MEK-1 caused an increase of ERK activity and inhibited STAT3((Tyr705)) phosphorylation. Conversely, inhibition of ERK activity by PD98059 reversed the R115777-induced inhibition of STAT3((Tyr705)) phosphorylation. R115777 also caused the inhibition of the binding of STAT3 to its consensus binding element. An increase in the activation of ERKs either by overexpressing MEK-1 or treatment of cells with R115777 caused an up-regulation in the levels of a cyclin-dependent kinase (cdk) inhibitor, p21(cip1/waf1). These observations suggest that R115777-induced growth inhibition is partly due to the prolonged activation of ERKs that mediates an inhibition of STAT3((Tyr705)) phosphorylation and an increase in the levels of p21(cip1/waf1) in human pancreatic adenocarcinoma cell lines.

Trichostatin A induces transforming growth factor beta type II receptor promoter activity and acetylation of Sp1 by recruitment of PCAF/p300 to a Sp1.NF-Y complex.

Transforming growth factor beta type II receptor (TbetaRII) is a tumor suppressor gene that can be transcriptionally silenced by histone deacetylases (HDACs) in cancer cells. In this report, we demonstrated the mechanism by which trichostatin A (TSA), an inhibitor of HDAC, induces the expression of TbetaRII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the TbetaRII promoter. This region of the TbetaRII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at -102 and -83, respectively). Treatment of cells with TSA activates the TbetaRII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1.NF-Y.HDAC complex that binds this DNA element. The recruitment of p300 and PCAF into the complex is associated with a concomitant acetylation of Sp1 and an overall decrease in the amount of HDAC associated with the complex. Transient overexpression of p300 or PCAF potentiated TSA-induced TbetaRII promoter activity. The effect of PCAF was dependent on its histone acetyltransferase activity, whereas that of p300 was independent. Stable transfection of PCAF caused an increase in TbetaRII mRNA expression, the association of PCAF with TbetaRII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the TbetaRII promoter through modulation of the components of a Sp1.NF-Y.p300.PCAF.HDAC-1 multiprotein complex. Moreover, the interaction of NF-Y with the Sp1-associated complex may further explain why this specific Sp1 site mediates transcriptional responsiveness to TSA.

Alterations of cell signaling pathways in pancreatic cancer.

Pancreatic ductal adenocarcinomas continue to have the worst prognosis of any adult malignancy with a five-year survival rate of less than 4%. One approach to improve patient survival from pancreatic cancer is to identify new biological targets that contribute to the aggressive pathogenecity of this disease and to develop reagents that will interfere with the function of these targets. Apart from the identification of the genetic profile of pancreatic cancer, a number of studies have focused on aberrant cell signaling pathways and their role in pancreatic cancer biology and response to therapy. This review, although not comprehensive, will discuss the salient features of several of these pathways. These include the roles of TGF beta signaling in both tumor suppression and tumor promotion and the effects of deregulation of phosphotyrosine kinase receptor signaling pathways in pancreatic cancer.

Requirement of a specific Sp1 site for histone deacetylase-mediated repression of transforming growth factor beta Type II receptor expression in human pancreatic cancer cells.

In this study, we demonstrate a novel mechanism by which down-regulation of transforming growth factor beta type II receptor (TbetaRII) is mediated by a histone deacetylase (HDAC) in pancreatic ductal adenocarcinoma (PDAC) cells. Treatment of PDAC cell lines BxPC-3 and MIA PaCa-2 with a specific HDAC inhibitor, trichostatin A (TSA), strongly activates TbetaRII promoter activity and induces TbetaRII expression. The transcriptional activation of TbetaRII by TSA was correlated with a decrease in HDAC activity and an increase in acetylated histone H4 protein. Correspondingly, an increase in the association of TbetaRII promoter with acetylated histone H4 was detected in the TSA-treated cells as determined by a chromatin immunoprecipitation assay. We found that a specific Sp1 site (Sp1C, located at -102 bp relative to the transcription start site) adjacent to an inverted CCAAT box (-83 bp) is required for TSA-mediated activation of the TbetaRII promoter. Furthermore, we determined that HDAC1 complexed with Sp1 in PDAC cells and that TSA treatment interfered with this association. Diminished binding of HDAC1 to the -112 to -65 bp region of the TbetaRII promoter after TSA treatment was confirmed by a DNA affinity precipitation assay. This is the first study to demonstrate the requirement of a specific Sp1 site for TSA-mediated transcriptional activation of TbetaRII. This study further suggests that the specificity of this Sp1 site for HDAC-mediated repression of TbetaRII may involve the interaction of the Sp1-HDAC1 complex with components of the cognate transcriptional regulators that bind to the inverted CCAAT box.

Rap1 reverses transcriptional repression of TGF-beta type II receptor by a mechanism involving AP-1 in the human pancreatic cancer cell line, UK Pan-1.

The TGF-beta signaling pathway has potent anti-mitogenic effects in epithelial cells and loss of negative growth regulation is often associated with increased tumorigenicity. The human pancreatic ductal adenocarcinoma cell line, UK Pan-1, which expresses DPC4, is not highly responsive to TGF-beta due to transcriptional repression of TGF-beta type II receptor (RII). Here, we show that UK Pan-1 cells transfected with a plasmid to overexpress rap1 protein (UK/rap1) causes an increase in RII transcription and restores sensitivity to TGF-beta growth inhibition. The overexpression of rap1 was associated with diminished ras signaling as measured by ras binding domain (RBD)-binding assays. Electrophoretic mobility shift assays (EMSA) analysis revealed increased binding of nuclear proteins to a previously identified positive regulatory element (PRE1) of the RII promoter in rap1 transfected cells. Competition with an oligo containing the AP-1 consensus site was able to inhibit this binding of nuclear proteins to the PRE1 region. Further EMSA analysis using antibodies to various AP-1 components revealed that junB antibodies partially depleted the increase in binding to the PRE1 seen in UK/rap1 cells while antibodies to other AP-1 constituents such as c-jun, c-fos, and ATF-1 had no effect on binding. Consistent with this data, transient transfection of UK Pan-1 cells with junB resulted in greater RII transcription (twofold) as measured by RII-luciferase assay. Mutation of the AP-1 site inhibited junB-mediated or rap1-mediated increases in RII promoter activity. These data suggest that rap1 signaling may mediate an increase in RII transcription via increased binding of nuclear factors including junB to the PRE1 region of the RII promoter.

Restoration of transforming growth factor-beta signaling enhances radiosensitivity by altering the Bcl-2/Bax ratio in the p53 mutant pancreatic cancer cell line MIA PaCa-2.

In this study, we investigated whether lack of transforming growth factor beta (TGF-beta) type II receptor (RII) expression and loss of TGF-beta signaling played a role in radiation resistance of pancreatic cancer cells MIA PaCa-2 that possess a mutated p53 gene. Transfection of this cell line with a RII cDNA led to a stimulation of the transcriptional activity of p3TP-Lux, a TGF-beta-responsive reporter construct. The RII transfectants (MIA PaCa-2/RII) showed a significant increase in sensitivity to radiation when compared with MIA PaCa-2/vector cells. The increase in sensitivity to radiation was reversed by neutralizing antibodies to TGF-beta, indicating that these changes were dependent on TGF-beta signaling. Compared with MIA PaCa-2/vector cells, MIA PaCa-2/RII cells showed a greater than 3-fold increase in apoptosis after radiation. Enhanced radiation sensitivity of MIA PaCa-2/RII cells was associated with an induction of Bax mRNA and protein that was followed by a release of cytochrome c and activation of caspase-3 and poly(ADP-ribose) polymerase cleavage after radiation exposure. Overexpression of Bcl-x(L) or treatment with antisense oligodeoxynucleotides targeted against Bax significantly inhibited radiation-induced apoptosis in MIA PaCa-2/RII but not in MIA PaCa-2/Vector cells, suggesting that Bax induction is necessary for radiation-induced TGF-beta signaling-mediated apoptosis. Thus, restoration of TGF-beta signaling sensitized these cells to ionizing radiation, although these cells possess a mutated p53 gene. In addition, disruption of RII function by dominant negative mutant of RII inhibited the radiation-induced TGF-beta signaling and apoptosis in primary cultures of mouse embryonic fibroblasts. Together, these observations imply that RII is an important component of radiation-induced TGF-beta signaling, and loss of function of RII may enhance resistance to radiation-induced apoptosis.