Gemcitabine resistance of pancreatic cancer cells is mediated by IGF1R dependent upregulation of CD44 expression and isoform switching.

Chemoresistance in pancreatic cancer cells may be caused by the expansion of inherently resistant cancer cells or by the adaptive plasticity of initially sensitive cancer cells. We investigated how CD44 isoforms switching contributed to gemcitabine resistance. Treating CD44 null/low single-cell clones with increasing amounts of gemcitabine caused an increase in expression of CD44 and development of gemcitabine resistant (GR) cells. Drug sensitivity, invasiveness, and EMT process was evaluated by MTT, Matrigel invasion assays, and western blots. Genetic knockdown and pharmacological inhibitors were used to examine the roles of CD44 and IGF1R in mediating gemcitabine resistance. CD44 promoter activity and its interactive EMT-related transcription factors were evaluated by luciferase reporter assay and chromatin immunoprecipitation assay. Kaplan-Meier curve was created by log-rank test to reveal the clinical relevance of CD44 and IGF1R expression in patients. We found silence of CD44 in GR cells partially restored E-cadherin expression, reduced ZEB1 expression, and increased drug sensitivity. The gemcitabine-induced CD44 expressing and isoform switching were associated with an increase in nuclear accumulation of phosphor-cJun, Ets1, and Egr1 and binding of these transcription factors to the CD44 promoter. Gemcitabine treatment induced phosphorylation of IGF1R and increased the expression of phosphor-cJun, Ets1, and Egr1 within 72 h. Stimulation or suppression of IGF1R signaling or its downstream target promoted or blocked CD44 promoter activity. Clinically, patients whose tumors expressed high levels of CD44/IGF1R showed a poor prognosis. This study suggests that IGF1R-dependent CD44 isoform switching confers pancreatic cancer cells to undergo an adaptive change in response to gemcitabine and provides the basis for improved targeted therapy of pancreatic cancer.

The biology and role of CD44 in cancer progression: therapeutic implications.

CD44, a non-kinase transmembrane glycoprotein, is overexpressed in several cell types including cancer stem cells and frequently shows alternative spliced variants that are thought to play a role in cancer development and progression. Hyaluronan, the main ligand for CD44, binds to and activates CD44 resulting in activation of cell signaling pathways that induces cell proliferation, increases cell survival, modulates cytoskeletal changes, and enhances cellular motility. The different functional roles of CD44 standard (CD44s) and specific CD44 variant (CD44v) isoforms are not fully understood. CD44v contain additional peptide motifs that can interact with and sequester growth factors and cytokines at the cell surface thereby functioning as coreceptors to facilitate cell signaling. Moreover, CD44v were expressed in metastasized tumors, whereas switching between CD44v and CD44s may play a role in regulating epithelial to mesenchymal transition (EMT) and in the adaptive plasticity of cancer cells. Here, we review current data on the structural and functional properties of CD44, the known roles for CD44 in tumorigencity, the regulation of CD44 expression, and the potential for targeting CD44 for cancer therapy.

Downregulation of STAT3/NF-κB potentiates gemcitabine activity in pancreatic cancer cells.

There is an unmet need to develop new agents or strategies against therapy resistant pancreatic cancer (PanCA). Recent studies from our laboratory showed that STAT3 negatively regulates NF-κB and that inhibition of this crosstalk using Nexrutine® (Nx) reduces transcriptional activity of COX-2. Inhibition of these molecular interactions impedes pancreatic cancer cell growth as well as reduces fibrosis in a preclinical animal model. Nx is an extract derived from the bark of Phellodendron amurense and has been utilized in traditional Chinese medicine as antidiarrheal, astringent, and anti-inflammatory agent for centuries. We hypothesized that "Nx-mediated inhibition of survival molecules like STAT3 and NF-κB in pancreatic cancer cells will improve the efficacy of the conventional chemotherapeutic agent, gemcitabine (GEM)." Therefore, we explored the utility of Nx, one of its active constituents berberine and its derivatives, to enhance the effects of GEM. Using multiple human pancreatic cancer cells we found that combination treatment with Nx and GEM resulted in significant alterations of proteins in the STAT3/NF-κB signaling axis culminating in growth inhibition in a synergistic manner. Furthermore, GEM resistant cells were more sensitive to Nx treatment than their parental GEM-sensitive cells. Interestingly, although berberine, the Nx active component used, and its derivatives were biologically active in GEM sensitive cells they did not potentiate GEM activity when used in combination. Taken together, these results suggest that the natural extract, Nx, but not its active component, berberine, has the potential to improve GEM sensitivity, perhaps by down regulating STAT3/NF-κB signaling. © 2016 Wiley Periodicals, Inc.

CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy.

PURPOSE: A subpopulation of pancreatic ductal adenocarcinoma (PDAC) cells is thought to be inherently resistant to chemotherapy or to give rise to tumor cells that become resistant during treatment. Here we determined the role of CD44 expression and its isoforms as a marker and potential target for tumor cells that give rise to invasive and gemcitabine-resistant tumors. EXPERIMENTAL DESIGN: RT-PCR, Western blotting, and DNA sequencing was used to determine CD44 isoform and expression levels. Flow cytometry was used to sort cells on the basis of their CD44 expression level. CD44 expression was knocked down using shRNA. Tumorigenic properties were determined by clonogenic and Matrigel assays, IHC, tumor growth in vivo using luciferase imaging and by tumor weight. RESULTS: We identified an invasive cell population that gives rise to gemcitabine-resistant tumors. These cancer cells express a high level of CD44 standard isoform and have an EMT phenotype (CD44s/EMT). In vivo, CD44s/EMT engraft and expand rapidly and give rise to tumors that express high levels of CD44 isoforms that contain multiple exon variants. CD44low-expressing cells show continued sensitivity to gemcitabine in vivo and knockdown of CD44 in CD44s/EMT cells increases sensitivity to gemcitabine and decreases invasiveness. CONCLUSIONS: PDAC cells expressing high levels of CD44s with a mesenchymal-like phenotype were highly invasive and developed gemcitabine resistance in vivo Thus, initial targeting CD44 or reversing the CD44high phenotype may improve therapeutic response. Clin Cancer Res; 22(22); 5592-604. ©2016 AACR.

Roles of c-Met and RON kinases in tumor progression and their potential as therapeutic targets.

c-Met and receptor originated from nantes (RON) are structurally related transmembrane phosphotyrosine kinase receptors. c-Met and RON show increased expression or activity in a variety of tumors leading to tumor progression and may play a role in acquired resistance to therapy. Although often co-expressed, the distinct functional roles of c-Met and RON are not fully understood. c-Met and RON form both activated homodimers and heterodimers with themselves and other families of phosphotyrosine kinase receptors. Inhibitors for c-Met and RON including small molecular weigh kinase inhibitors and neutralizing antibodies are in pre-clinical investigation and clinical trials. Several of the tyrosine kinase inhibitors have activity against both c-Met and RON kinases whereas the antibodies generally are target specific. As with many targeted agents used to treat solid tumors, it is likely that c-Met/RON inhibitors will have greater benefit when used in combination with chemotherapy or other targeted agents. A careful analysis of c-Met/RON expression or activity and a better elucidation of how they influence cell signaling will be useful in predicting which tumors respond best to these inhibitors as well as determining which agents can be used with these inhibitors for combined therapy.

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.

Cooperativity of oncogenic K-ras and downregulated p16/INK4A in human pancreatic tumorigenesis.

Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.

Combined targeting of STAT3/NF-κB/COX-2/EP4 for effective management of pancreatic cancer.

PURPOSE: Near equal rates of incidence and mortality emphasize the need for novel targeted approaches for better management of patients with pancreatic cancer. Inflammatory molecules NF-κB and STAT3 are overexpressed in pancreatic tumors. Inhibition of one protein allows cancer cells to survive using the other. The goal of this study is to determine whether targeting STAT3/NF-κB crosstalk with a natural product Nexrutine can inhibit inflammatory signaling in pancreatic cancer. EXPERIMENTAL DESIGN: HPNE, HPNE-Ras, BxPC3, Capan-2, MIA PaCa-2, and AsPC-1 cells were tested for growth, apoptosis, cyclooxygenase-2 (COX-2), NF-κB, and STAT3 level in response to Nexrutine treatment. Transient expression, gel shift, chromatin immunoprecipitation assay was used to examine transcriptional regulation of COX-2. STAT3 knockdown was used to decipher STAT3/NF-κB crosstalk. Histopathologic and immunoblotting evaluation was performed on BK5-COX-2 transgenic mice treated with Nexrutine. In vivo expression of prostaglandin receptor E-prostanoid 4 (EP4) was analyzed in a retrospective cohort of pancreatic tumors using a tissue microarray. RESULTS: Nexrutine treatment inhibited growth of pancreatic cancer cells through induction of apoptosis. Reduced levels and activity of STAT3, NF-κB, and their crosstalk led to transcriptional suppression of COX-2 and subsequent decreased levels of prostaglandin E2 (PGE2) and PGF2. STAT3 knockdown studies suggest STAT3 as negative regulator of NF-κB activation. Nexrutine intervention reduced the levels of NF-κB, STAT3, and fibrosis in vivo. Expression of prostaglandin receptor EP4 that is known to play a role in fibrosis was significantly elevated in human pancreatic tumors. CONCLUSIONS: Dual inhibition of STAT3-NF-κB by Nexrutine may overcome problems associated with inhibition of either pathway.

Oncogenic K-Ras and loss of Smad4 mediate invasion by activating an EGFR/NF-κB Axis that induces expression of MMP9 and uPA in human pancreas progenitor cells.

Activating K-Ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). To further study the individual and combinatorial roles of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by either expressing oncogenic K-Ras (HPNE/K-Ras), by shRNA knock down of Smad4 (HPNE/ShSmad4) or by creating both alterations in the same cell line (HPNE/K-Ras/ShSmad4). We previously found that expression of oncogenic K-Ras caused an increase in expression of EGFR and loss of Smad4 further enhanced the up regulation in expression of EGFR and that this increase in EGFR was sufficient to induce invasion. Here we further investigated the mechanism that links mutational alterations and EGFR expression with invasion. The increase in EGFR signaling was associated with up regulation of MMP9 and uPA protein and activity. Moreover, the increase in EGFR signaling promoted a nuclear translocation and binding of RelA (p65), a subunit of NF-κB, to the promoters of both MMP-9 and uPA. Treatment of HPNE/K-Ras/ShSmad4 cells with an inhibitor of EGFR reduced EGF-mediated NF-κB nuclear translocation and inhibitors of either EGFR or NF-κB reduced the increase in MMP-9 or uPA expression. In conclusion, this study provides the mechanism of how a combination of oncogenic K-Ras and loss of Smad4 causes invasion and provides the basis for new strategies to inhibit metastases.

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.

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion.

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.

Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the expression of EGFR and to promote invasion of immortalized human pancreas ductal cells.

Activating mutation of K-ras and inactivation of DPC4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). A separate common event in PDAC progression is increased expression of phosphotyrosine kinase receptors (PTKRs). In our study, we examined whether activating mutations of K-ras and loss of Smad4 play a role in causing the aberrant expression of PTKRs. Immortalized human pancreas ductal cells (HPNE) were genetically modified by expressing oncogenic K-ras and/or by shRNA knockdown of Smad4. EGFR and erbB2 protein levels but not Ron or IGF-1R were substantially upregulated in HPNE cells that express K-ras((GD12)). The increased expression of EGFR in HPNE cells that expressed K-ras((GD12)) was mediated by both stabilizing EGFR protein and by increasing EGFR transcription. TGF-beta signaling partially suppressed K-ras((GD12)) induced EGFR transcription in Smad4 intact HPNE cells; whereas knockdown of Smad4 in cells expressing K-ras((GD12)) further enhanced expression of EGFR and erbB2. The upregulation of EGFR and erbB2 was associated with an increase of invasion, which was blocked by a kinase inhibitor of EGFR. Our study indicates for the first time, that oncogenic ras and loss of Smad signaling cooperate to upregulate EGFR and erbB2, which plays a role in promoting invasion.

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.

Smad4-dependent TGF-beta signaling suppresses RON receptor tyrosine kinase-dependent motility and invasion of pancreatic cancer cells.

Transforming growth factorbeta (TGF-beta) signals through Smad-dependent and Smad-independent pathways. However, Smad signaling is altered by allelic deletion or intragenic mutation of the Smad4 gene in more than half of pancreatic ductal adenocarcinomas. We show here that loss of Smad4-dependent signaling leads to aberrant expression of RON, a phosphotyrosine kinase receptor, and that signaling by RON cooperates with Smad4-independent TGF-beta signaling to promote cell motility and invasion. Restoring Smad4 expression in a pancreatic ductal adenocarcinoma cell line that is deficient in Smad4 repressed RON expression. Conversely, small interference RNA knock down of Smad4 or blocking TGF-beta signaling with a TGF-beta type I receptor kinase inhibitor in Smad4-intact cell lines induced RON expression. TGF-beta-induced motility and invasion were inhibited in cells that express Smad4 and that have low levels of RON compared with isogenically matched cells that were deficient in Smad4. Furthermore, knocking down RON expression in Smad4-deficient cells suppressed TGF-beta-mediated motility and invasion. We further determined that Smad4-dependent signaling regulated RON expression at the transcriptional level by real-time reverse transcription PCR and RON promoter luciferase reporter assays. Functional inactivation by site-directed mutations of two Smad binding sites on the RON promoter inhibited TGF-beta-mediated repression of RON promoter activity. These studies indicate that loss of Smad4 contributes to aberrant RON expression and that cross-talk of Smad4-independent TGF-beta signaling and the RON pathway promotes an invasive phenotype.

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.

Autocrine-mediated ErbB-2 kinase activation of STAT3 is required for growth factor independence of pancreatic cancer cell lines.

Pancreatic ductal adenocarcinoma (PDAC) cell lines, MIA PaCa-2, and UK Pan-1, were used to investigate the role of ErbB2 in PDAC oncogenesis. Both these cell lines exhibit exogenous growth factor-independent proliferation that was attributed to the production of autocrine growth factors and/or overexpression of growth factor receptors. The exogenous growth factor-independent phenotype displayed by these PDAC cell lines was dependent on ErbB2 kinase activity since treatment of cells with tyrphostin AG879 prevented serum-free media (SFM) induction of cell proliferation. We determined that ErbB2 kinase contributed to aberrant cell cycle regulation in PDAC through the induction of cyclin D1 levels and the suppression of p21(Cip1) and p27(Kip1). Inhibition of ErbB2 kinase led to cell cycle arrest marked by an increased association of p27(Kip1) with cdk2 and reduced levels of phosphorylated pRb. We further observed constitutive STAT3 activation in the PDAC cell lines and an increase in STAT3 activation upon stimulating quiescent cells with SFM. Inhibitors of ErbB2 kinase blocked STAT3 activation, whereas inhibition of EGFR kinase led to a slight reduction of STAT3 activation. STAT3 was coimmunoprecipitated with ErbB2. SFM stimulation caused an increase in the association of ErbB2 and STAT3, which was blocked by inhibition of ErbB2 kinase. Expression of a STAT3 dominant negative prevented SFM-stimulated cell proliferation of MIA PaCa-2 cells, suggesting that activation of STAT3 by ErbB2 is required for a growth factor-independent phenotype of these cells. Consistent with this observation in PDAC cell lines, we found that most PDAC tumor specimens (10 of 11) showed constitutive activation of STAT3 and that ErbB2 was readily detected in most of these tumors (nine of 11). We believe that these findings indicate a novel mechanism of oncogenesis in PDAC and may suggest future therapeutic strategies in the treatment of PDAC.

Acetylated sp3 is a transcriptional activator.

Sp3 transcription factor can either activate or repress target gene expression. However, the molecular event that controls this dual function is unclear. We previously reported (Ammanamanchi, S., and Brattain, M. G. (2001) J. Biol. Chem. 276, 3348-3352) that unmodified Sp3 acts as a transcriptional repressor of transforming growth factor-beta receptors in MCF-7L breast cancer cells. We now report that histone deacetylase inhibitor trichostatin A (TSA) induces acetylation of Sp3, which acts as a transcriptional activator of transforming growth factor-beta receptor type II (RII) in MCF-7L cells. Mutation analysis indicated the TSA response is mediated through a GC box located on the RII promoter, which was previously identified as an Sp1/Sp3-binding site that was critical for RII promoter activity. Ectopic Sp3 expression in Sp3-deficient MCF-7E breast cancer cells repressed RII promoter activity in the absence of TSA. However, in the TSA-treated MCF-7E cells ectopic Sp3 activated RII promoter. Histone acetyltransferase p300 was shown to acetylate Sp3. Sp3-mediated RII promoter activity was stimulated by wild type p300 but not the histone acetyltransferase domain-deleted mutant p300 in MCF-7L cells, suggesting the positive effect of p300 acetylase activity on Sp3. Consequently, the results presented in this manuscript demonstrate that acetylation acts as a switch that controls the repressor and activator role of Sp3.