Sp1 is required for transforming growth factor-beta-induced mesenchymal transition and migration in pancreatic cancer cells.

Transition from a sessile epithelial phenotype to a migrating mesenchymal phenotype is a crucial step in transforming growth factor-beta (TGF-beta)-induced pancreatic cancer cell migration and invasion. These profound morphologic and functional alterations are associated with characteristic changes in TGF-beta-regulated gene expression, defined by rapid repression of epithelial markers and a strong and sustained transcriptional induction of mesenchymal markers such as the intermediate filament vimentin. In this study, we have analyzed the role of the transcription factor Sp1 in TGF-beta-induced and Smad-mediated gene regulation during epithelial to mesenchymal transition (EMT) and migration of pancreatic cancer cells. Here, we show that Sp1 is required for TGF-beta-induced EMT, and that this function is especially mediated through transcriptional induction of vimentin. Our results emphasize the functional relevance of vimentin in TGF-beta-induced EMT because prevention of its induction strongly reduces cell migration. Altogether, this study helps to better understand the role of Sp1 in TGF-beta-induced progression of pancreatic cancer. It suggests that Sp1, via transcriptional induction of vimentin, cooperates with activated Smad complexes in mesenchymal transition and migration of pancreatic cancer cells upon TGF-beta stimulation.

The tumor suppressor KLF11 mediates a novel mechanism in transforming growth factor beta-induced growth inhibition that is inactivated in pancreatic cancer.

c-myc promoter silencing is a key step in epithelial cell growth inhibition by transforming growth factor beta (TGFbeta). During carcinogenesis, however, epithelial cells escape from c-myc repression and consequently become refractory to TGFbeta-mediated antiproliferation. Here, we assessed the role of the repressor, KLF11, in TGFbeta-induced growth inhibition in normal epithelial as well as pancreatic carcinoma cells. Endogenous KLF11 was stably down-regulated by RNA interference technology, and the functional consequences were studied by proliferation assays, reporter assays, DNA binding studies, and expression analyses. Coimmunoprecipitation and glutathione S-transferase pulldown assays were conducted to define KLF11-Smad3 interaction and U0126 was administered to examine the effects of the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase on complex formation and c-myc promoter binding of KLF11 and Smad3 in pancreatic cancer cells. In TGFbeta-stimulated normal epithelial cells, nuclear KLF11, in concert with Smad3, binds to and represses transcription from the core region of the TGFbeta-inhibitory element (TIE) of the c-myc promoter. Disruption of KLF11-Smad3 interaction or small interfering RNA-mediated knockdown of endogenous KLF11 strongly diminishes Smad3-TIE promoter binding and repression, and consequently impairs TGFbeta-mediated growth inhibition. In pancreatic cancer cells with oncogenic Ras mutations, hyperactive ERK counteracts TGFbeta-induced c-myc repression and growth inhibition through at least two mechanisms, i.e., via disruption of KLF11-Smad3 complex formation and through inhibition of KLF11-Smad3 binding to the TIE element. Together, these results suggest a central role for KLF11 in TGFbeta-induced c-myc repression and antiproliferation and identifies a novel mechanism through which ERK signaling antagonizes the tumor suppressor activities of TGFbeta in pancreatic cancer cells with oncogenic Ras mutations.

TGFbeta-regulated transcriptional mechanisms in cancer.

Transforming growth factor-beta (TGFbeta) has been implicated in oncogenesis for many years. The multifunctional activities of TGFbeta endow it with both tumor suppressor and tumor promoting activities, depending on the stage of carcinogenesis and the responsivity of the tumor cell. In early tumor stages, TGFbeta inhibits epithelial cell growth through induction of apoptosis and cell cycle arrest. During tumor development, however, many tumor cells lose their growth-inhibitory responses to TGFbeta owing to genetic alterations or signaling perturbations such as oncogenic Ras signaling. Loss of TGFbeta-growth inhibition is commonly associated with increased tumor cell invasion and metastasis of tumor cells that undergo an epithelial-mesenchymal transition. Interestingly, the tumor-promoting effects of TGFbeta on the tumor cells are observed particularly in cells in which TGFbeta-signaling remains functional despite loss of growth control by TGFbeta. New insights into transcriptional mechanisms activated by TGFbeta are providing a better understanding of the cellular changes involved in the switch of TGFbeta from a tumor suppressor to a tumor promotor.

Smad-Sp1 complexes mediate TGFbeta-induced early transcription of oncogenic Smad7 in pancreatic cancer cells.

The transcription factor Sp1 has been implicated in cell-type-specific activation of transforming growth factor-beta (TGFbeta) target genes in normal epithelial cells as well as in aberrant gene activation by TGFbeta in epithelial tumor cells. Here, we have examined the interaction of Sp1 with components of the Smad signaling cascade and its role in TGFbeta-induced early gene expression in pancreatic cancer cells. Gene expression profiling was carried out in mithramycin-A-treated cells to identify Sp1-regulated TGFbeta early response genes. We found that in pancreatic cancer cells Smad proteins and Sp1 cooperatively regulate expression of a distinct set of TGFbeta target genes potentially involved in tumor progression, including MMP-11, cyclin D1 and Smad7. Mechanistically, TGFbeta rapidly induces nuclear translocation of Smad proteins and subsequently stimulates Smad-Sp1 complex formation. Using the Smad7 promoter as a model for Smad-/Sp1-induced early gene activation, we demonstrated that this interaction increases Sp1 binding to GC-rich promoter boxes and results in superinduction of Sp1-mediated transcription. Moreover, inhibition of Sp1-DNA binding or transfection of Sp1-specific siRNA prevents TGFbeta-induced Smad7 expression and consequently enhances Smad signaling in pancreatic cancer cells, as indicated by increased receptor-mediated phosphorylation of Smad3. We thus conclude that Sp1 strongly contributes to the aberrant transcriptional response of transformed epithelial cells to TGFbeta stimulation.

Blockage of intermediate-conductance Ca2+-activated K+ channels inhibit human pancreatic cancer cell growth in vitro.

Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (K-ras G12D (+/-), p53 R273C, Deltap16), BxPC-3 (smad4-, p53 Y220C, Deltap16), and MiaPaCa-2 [transforming growth factor-beta receptor type II defect, K-ras G12C(-/-), p53 R248W, Deltap16] human pancreatic cancer cell lines. In BxPC-3 and the MiaPaCa-2 cells, we could identify approximately 600 or approximately 1200 functional Ca2+-activated K+ channels (IK) per cell, respectively, whereas PANC-1 cells expressed approximately 200 functional IK channels per cell. These channels were observed by using pipette solutions buffering [Ca2+]i to 1 microM. The channels were voltage-independent, blocked by charybdotoxin, clotrimazole, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), and blocked by Ba2+ in a voltage-dependent manner. In the presence of 10 microM clotrimazole or TRAM-34, proliferation of the BxPC-3 as well as the MiaPaCa-2 cells was completely stopped. In contrast, proliferation of PANC-1 cells was hardly affected by clotrimazole or TRAM-34. Proliferation in all three cell lines could be inhibited in the presence of the Ca2+ channel antagonists verapamil, diltiazem, and nifedipine. By quantitative RT-PCR, we could show that MiaPaCa-2 cells exhibit a 2.8-fold and BxPC3 cells a more than 8-fold elevated level of IK mRNA level compared with PANC-1 cells. Interestingly, in primary pancreatic tumors we found a tremendous up-regulation of IK mRNA. In eight of nine (or 89%) primary pancreatic tumor tissues, we found a 6- to 66-fold increase in IK mRNA. Our findings suggest that a certain amount of functional IK channels is crucial for the proliferation of some pancreatic cancer types. The blockade of IK channels may ultimately prove useful as a therapeutic option for some patients with ductal adenocarcinoma of the pancreas with an up-regulated IK channel expression.

KLF11 mediates a critical mechanism in TGF-beta signaling that is inactivated by Erk-MAPK in pancreatic cancer cells.

BACKGROUND & AIMS: Smad-regulated transcription plays a central role in transforming growth factor (TGF)-beta-induced cell growth inhibition and tumor suppression. Like the Smads, KLF11 is an early response transcription factor that mediates TGF-beta-induced growth inhibition in untransformed epithelial cells. Here, we investigated the functional implications of KLF11 in TGF-beta signaling and transcription in normal epithelial as well as pancreatic cancer cells. METHODS: The effects of KLF11 on TGF-beta signaling and transcription were examined on the levels of reporter transactivation, Smad2 phosphorylation, and expression of endogenous TGF-beta-regulated genes. Promoter analysis, real-time polymerase chain reaction, and coimmunoprecipitation studies were performed to study KLF11-induced and mSin3A corepressor-mediated repression of Smad7. Erk-induced KLF11 phosphorylation was examined in vitro and in vivo, and its impact on KLF11-mSin3A-mediated Smad7 repression was verified in pancreatic cancer cells using site-directed mutagenesis. RESULTS: KLF11 potentiates TGF-beta signaling by terminating the inhibitory Smad7 loop. Mechanistically, KLF11 represses TGF-beta-induced transcription from the Smad7 promoter by recruiting mSin3a via GC-rich sites. This function is inhibited in pancreatic cancer cells with oncogenic Ras mutations, in which Erk/mitogen-activated protein kinase phosphorylates KLF11, leading to disruption of KLF11-mSin3a interaction. Expression of an Erk-insensitive KLF11 mutant restores both mSin3a binding and Smad7 repression and results in enhanced TGF-beta signaling in pancreatic cancer cells. CONCLUSIONS: These results define a novel mechanism in TGF-beta-regulated gene expression. KLF11 potentiates Smad-signaling activity in normal epithelial cells through termination of the negative feedback loop imposed by Smad7. The fact that this function of KLF11 is inhibited by oncogenic Erk/mitogen-activated protein kinase in pancreatic cancer cells emphasizes the importance of this mechanism for oncogenesis.

Expression profiling of the influence of RAS mutants on the TGFB1-induced phenotype of the pancreatic cancer cell line PANC-1.

Expression profiling analyses were used to elucidate the functional relevance of RAS proteins in mediating the effect of TGFB1 on the transcriptional phenotype of the pancreatic cancer cell line PANC-1. Despite the presence of one mutated KRAS2 allele in parental PANC-1 pancreatic cancer cells, RAS-dependent signal transduction remained susceptible to stimulation by EGF and TGFB1. To analyze the impact of RAS proteins on the TGFB1-induced transcriptional phenotype, we used PANC-1 cells stably transfected with a dominant negative HRAS(S17N) mutant or with a constitutively active KRAS2(G12V) mutant. TGFB1 treatment of mock-transfected PANC-1 cells led to an expression profile suggestive of epithelial-mesenchymal transdifferentiation (EMT). Profiling of the HRAS(S17N)-expressing clone demonstrated that induction of endogenous RAS activity by TGFB1 is required for the development of the TGFB1-induced transcriptional phenotype of PANC-1 cells. The expression of the KRAS2(G12V) mutant by itself repressed transcription of markers of epithelial differentiation and induced transcription of several extracellular matrix-associated genes. This effect was not enhanced further by TGFB1 treatment. In contrast, transcript levels of genes associated with proliferation and cell cycle progression did not appear to be the primary targets of the synergism between the RAS- and TGFB1-dependent cascades. The introduction of the dominant negative and the constitutively active RAS mutants induced partly overlapping and partly inverse effects on the TGFB1-induced expression profile of PANC-1 cells. Additional mechanisms such as the induction of autocrine loops and the use of different RAS isoforms or alternate, ERK-independent signaling pathways may be involved in the interaction between the RAS- and the TGFB1-dependent signaling cascades.