Transforming growth factor ␤ type II receptor (T␤RII) 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 T␤RII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the T␤RII promoter. This region of the T␤RII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at ؊102 and ؊83, respectively). Treatment of cells with TSA activates the T␤RII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1⅐NF-Y⅐HDAC 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 T␤RII 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 T␤RII mRNA expression, the association of PCAF with T␤RII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the T␤RII promoter through modulation of the components of a Sp1⅐NF-Y⅐p300⅐PCAF⅐HDAC-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. TGF-␤1 plays a significant role in the growth inhibition of most normal epithelial and some cancer cells (1). TGF-␤ mediates its biological effects through cell surface receptors known as TGF-␤ type I receptor (T␤RI) and TGF-␤ type II receptor (T␤RII). Its intracellular signaling is initiated upon the selective binding of the active cytokine to the T␤RII homodimer. T␤RII is a ubiquitously expressed and constitutively active serine/threonine kinase. Ligand binding to T␤RII induces the assembly of a heterotetrameric complex consisting of T␤RI and T␤RII. Once the receptor complex is formed, T␤RII phosphorylates and thereby activates the T␤RI serine/threonine kinase. Activation of T␤RI propagates downstream signaling via Smad family proteins. T␤RI directly interacts with and phosphorylates Smad2 and Smad3. These Smads bind Smad4 and then result in the translocation of this complex to the nucleus and modulate TGF-␤-responsive gene expression (2-4).The TGF-␤ signaling pathway is inactivated in many tumors. Loss of negative growth regulation by TGF-␤ affords cells a selective growth advantage associated with decreased dependence of exogenous growth factor and increased tumorigenicity. Frequently, inhibition of TGF-␤ signaling occurs by either abolition of the function of a common mediator, Smad4, or interference with T␤RII function (5, 6). Smad4 and T␤RII are tumor suppressor gene...

The role of Smad4 in transforming growth factor B (TGFB)-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 TGFBmediated EMT as determined by increased expression of vimentin and decreased expression of B-catenin and E-cadherin. TGFB-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, TGFB inhibited STAT3 Tyr705 phosphorylation in Smad4-intact cells. The decrease in STAT3Tyr705 phosphorylation was linked to a TGFB/Smad4-dependent and enhanced activation of extracellular signalregulated 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 TGFBinduced 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 TGFB-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 TGFB from a tumor-suppressive to a tumor-promoting pathway in pancreatic cancer. [Cancer Res 2008;68(11):4221-8]

The tumor suppressor protein p53 is a pivotal regulator of apoptosis, and prostate cancer cells that lack p53 protein are moderately resistant to apoptotic death by ionizing radiation. Genes encoding the transcription factor early growth response-1 (EGR-1) and cytokine tumor necrosis factor-␣ (TNF-␣) were induced upon irradiation of prostate cancer cells, and inhibition of EGR-1 function resulted in abrogation of both TNF-␣ induction and apoptosis. Induction of the TNF-␣ gene by ionizing radiation and EGR-1 was mediated via a GC-rich EGR-1-binding motif in the TNF-␣ promoter. Because TNF-␣ induces apoptosis in prostate cancer cells, these findings suggest that, in the absence of p53, ionizing radiation-inducible apoptosis is mediated by EGR-1 via TNF-␣ transactivation.

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In this study, we investigated whether lack of transforming growth factor ␤ (TGF-␤) type II receptor (RII) expression and loss of TGF-␤ 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-␤؊respon-sive 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-␤, indicating that these changes were dependent on TGF-␤ signaling.