The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor B (TGF-B) plays a key role in cancer metastasis, signaling through the TGF-B type I/II receptors (TBRI/II). We hypothesized that targeting TBRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-B1Àinduced cell migration and invasion, and induced anoikis. In vivo , LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TBRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TBRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis. [Mol Cancer Ther 2008;7(4):829 -40]

Transcription factor nuclear factor-κB (NF-κB) is constitutively activated in most pancreatic cancer tissues and cell lines but not in normal pancreas nor in immortalized/nontumorigenic human pancreatic ductal epithelial cells. Inhibition of constitutive NF-κB activation in pancreatic cancer cell lines suppresses tumorigenesis and tumor metastasis. Recently, we identified autocrine secretion of proinflammatory cytokine interleukin (IL)-1α as the mechanism of constitutive NF-κB activation in metastatic pancreatic cancer cell lines. However, the role of IL-1α in determining the metastatic potential of pancreatic tumor remains to be further investigated. In the current study, we stably expressed IL-1α in the nonmetastatic, IL-1α-negative MiaPaCa-2 cell lines. Our results showed that the secretion of IL-1α in MiaPaCa-2 cells constitutively activated NF-κB and increased the expression of NF-κB downstream genes involved in the different steps of the metastatic cascade, such as urokinase-type plasminogen activator, vascular endothelial growth factor, and IL-8. MiaPaCa-2/IL-1α cells showed an enhanced cell invasion in vitro compared with parental MiaPaCa-2 cells and induced liver metastasis in an orthotopic mouse model. The metastatic phenotype induced by IL-1α was inhibited by the expression of phosphorylationdefective IκB (IκB S32, 36A), which blocked NF-κB activation. Consistently, silencing the expression of IL-1α by short hairpin RNA in the highly metastatic L3.6pl pancreatic cancer cells completely suppressed their metastatic spread. In summary, these findings showed that IL-1α plays key roles in pancreatic cancer metastatic behavior through the constitutive activation of NF-κB. Our findings further support the possible link between inflammation and cancer and suggest that IL-1α may be a potential therapeutic target for treating pancreatic adenocarcinoma.Requests for reprints: Paul J. Chiao,

Purpose:To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA. Experimental Design:We hypothesized that targeting the 3 ¶-untranslated region of Gli-1mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3 ¶-untranslated region Gli-1sequence of high GU content. Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1 + ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA. Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.

BACKGROUNDConstitutive activation of nuclear factor‐κB (NF‐κB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF‐κB activity plays a key role in the aggressive behavior of this disease. In an attempt to identify an effective therapeutic agent for pancreatic cancer, the authors studied the role of FUT‐175, a synthetic serine protease inhibitor, in the inhibition of NF‐κB activation and the induction of apoptotic responses.METHODSTo examine the effect of FUT‐175 on the inhibition of NF‐κB and the induction of apoptosis in pancreatic cancer cell lines, Western and Northern blot analyses, electromobility shift (EMSA), luciferase reporter gene, DNA fragmentation, immunoprecipitation, in vitro kinase, small interfering RNA (siRNA), and chromatin immunoprecipitation (ChIP) assays were performed.RESULTSIn a time‐dependent and dose‐dependent manner, FUT‐175 inhibited IκBα phosphorylation and NF‐κB activation, thereby inhibiting the antiapoptotic activity of NF‐κB. Simultaneously, FUT‐175 up‐regulated the expression of tumor necrosis factor receptor‐1 (TNFR1), which in turn activated the proapoptotic caspase‐8 and Bid pathways and induced apoptosis in pancreatic cancer cells. FUT‐175‐induced activation of Fas‐associated death domain (FADD) and caspase‐8 was suppressed by RNA interference‐mediated inhibition of TNFR1 expression. Furthermore, expression of the transcription factor PEA3 was up‐regulated by FUT‐175 and was involved in FUT‐175–mediated TNFR1 expression.CONCLUSIONSThese results suggested a possible mechanism by which FUT‐175 may disrupt interconnected signaling pathways by both suppressing the NF‐κB antiapoptotic activity and inducing TNFR‐mediated apoptosis. Supported by this unique function as a NF‐κB inhibitor and apoptosis inducer, this well‐established synthetic serine protease inhibitor with as‐of‐yet poorly understood mechanisms of actions appears to be a potentially therapeutic agent for pancreatic cancer. Cancer 2007. © 2007 American Cancer Society.

A 53-year-old man who had had an anal fistula for 20 years was admitted to our hospital with a large intestinal obstruction. Barium enema and colonoscopy confirmed advanced rectal cancer and we palpated a soft tumor, 3 cm in diameter, with inflammatory induration on the right side of the rectum. After draining a perianal abscess caused by the anal fistula, we performed low anterior resection. Histological examination of the perianal necrotic tissue obtained during resection of the perianal tumor encompassing the anal fistula revealed adenocarcinoma. Since the histology of the perianal lesion was identical to that of the rectal cancer, a diagnosis of cancer implantation rather than carcinoma originating in the anal fistula was entertained. Although the recurrence of rectal cancer by mucosal implantation is not uncommon, the coincidental implantation of rectal cancer in an anal fistula is extremely rare.

No abstract

A 72-year-old Japanese man had a history of proximal gastrectomy for early gastric cancer located in the upper third of the stomach in 2007. Our usual treatment strategy for early gastric cancer in the upper third of the stomach in 2007 was open proximal gastrectomy reconstructing by jejunal interposition with a 10 cm single loop. Upper gastrointestinal fiberscopy for annual follow-up revealed a type 0-IIc-shaped tumor with ulcer scar, 4.0 cm in size, located in the gastric remnant near the jejunogastrostomy. A clinical diagnosis of cancer of the gastric remnant, clinical T1b(SM)N0M0, Stage IA, following the proximal gastrectomy was made and a laparoscopic approach was selected because of the cancer's early stage. Remnant total gastrectomy with D1 plus lymphadenectomy was carried out with five ports by a pneumoperitoneal method. Complete resection of the reconstructed jejunum was undergone along with the jejunal mesentery. Reconstruction by the Roux-en-Y method via the antecolic route was selected. Total operative time was 395 min and blood loss was 40 mL. Our patient was the first successful case of resection for carcinoma of the gastric remnant following proximal gastrectomy reconstructed with jejunal interposition in a laparoscopic approach.