Takao Ohtsuka

Intraductal papillary-mucinous tumor of the pancreas concomitant with ductal carcinoma of the pancreas

Background: Despite the recent progress of diagnostic and therapeutic modalities, the clinical course of patients with ductal carcinoma (DC) of the pancreas remains dismal. Intraductal papillary-mucinous tumor of the pancreas (IPMT) is sometimes accompanied by malignant diseases of the other organs and pancreatic adenocarcinoma. Thus, IPMT may be a potential diagnostic clue to DC of the pancreas at early phase. Methods: Clinicopathologic findings of 7 Japanese patients with IPMT of the pancreas concomitant with independent DC were examined and compared with those of 69 patients with IPMT alone and of 70 with DC alone. Results: The seven patients corresponded to 9.2% of 76 patients with IPMT and 9.1% of 77 patients with DC. The seven male patients ranged from 55 to 75 years with a mean of 64.3. DC was synchronous with IPMT in five patients, metachronous to IPMT (4 years after IPMT) in one, and synchronous with IPMT and metachronous to IPMT and DC (7 years after IPMT) in the other. In 4 patients, the presence of IPMT led to the diagnosis of DC. All the 7 IPMTs were of branch type with a mean diameter of 3.0 cm. The IPMT was located in the head of the pancreas in 3, body in 2 and tail in the other 2. All the 7 IPMTs were adenoma with mild dysplasia. Two of the 7 patients with DC had in situ carcinoma, 1 minimally invasive carcinoma and the remaining 4 invasive carcinoma. The mean diameter of seven DCs (3.0 cm) with IPMT were smaller than that of 70 DCs (3.6 cm) (8P = 0.0295). Stage (stage I/II/III/IV = 3/0/3/1) of the seven DCs concomitant with IPMT were significantly earlier than that (stage I/II/III/IV = 5/6/28/31) of the other 70 (p = 0.0203). The survival curve of the 7 patients with IPMT and DC was significantly better than that of the 70 with DC alone (p = 0.0460). Conclusions: Clinicians should pay attention to the possible presence of DC of the pancreas in male patients with intraductal papillary-mucinous adenoma of the pancreas of branch type in their 6th to 8th decades.

ASC is a Bax adaptor and regulates the p53¿Bax mitochondrial apoptosis pathway

The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein–protein interaction domain. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53–Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53–Bax network.

A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in the p53-mediated apoptosis pathway.

We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502–802) interacted with the IFI16 Pyrin domain (aa 1–130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNA-damaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.

Tumor-stromal cell interaction under hypoxia increases the invasiveness of pancreatic cancer cells through the hepatocyte growth factor/c-Met pathway.

The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF‐1α expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP‐2, MMP‐7, MT1‐MMP and c‐Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c‐Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c‐Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF‐1α expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF‐1α expression but also the c‐Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c‐Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

Invasive carcinoma derived from the nonintestinal type intraductal papillary mucinous neoplasm of the pancreas has a poorer prognosis than that derived from the intestinal type.

BACKGROUND Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is divided into 4 subtypes: an intestinal type, a gastric type, a pancreatobiliary type, and an oncocytic type. The purposes of this study were to clarify the outcomes and the characteristics of invasive carcinoma derived from IPMN (invasive IPMC) by focusing on these subtypes with a comparison to conventional invasive ductal carcinoma (IDC) of the pancreas. METHODS A total of 30 patients with invasive IPMC were reviewed, and the tumors were divided into 2 pathologic subtypes, intestinal and nonintestinal type. The prognosis and characteristics of the 2 subtypes were evaluated. Furthermore, the prognosis of 119 patients with conventional IDC was compared with that of patients with invasive carcinoma derived from the intestinal or nonintestinal type IPMN. RESULTS The 5-year survival rate of patients with the nonintestinal type (0.0%) was as poor as that of patients with conventional IDC (19.9%; P = .67). The patients with the intestinal type (66.7%) had a more favorable prognosis than patients with conventional IDC (P < .001). The nonintestinal type was characterized by positive lymphatic invasion and tubular invasive pattern. CONCLUSION Invasive carcinoma derived from the nonintestinal type IPMN characterized by lymphatic invasion and tubular invasive pattern is associated with a poor prognosis.

GAMT, a p53-Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress

The p53 tumor suppressor protein has a well-established role in cell-fate decision-making processes. However, recent discoveries indicate that p53 has a non-tumor-suppressive role. Here we identify guanidinoacetate methyltransferase (GAMT), an enzyme involved in creatine synthesis, as a p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53-dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53-->GAMT upregulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP-generating energy source. These results highlight that p53-dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. The p53-->GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism.

Follow-up study after resection of intraductal papillary mucinous neoplasm of the pancreas; special references to the multifocal lesions and development of ductal carcinoma in the remnant pancreas

BACKGROUND Frequency and characteristics of metachronous occurrence of multifocal intraductal papillary mucinous neoplasms (IPMNs) or distinct pancreatic ductal adenocarcinomas (PDACs) in the remnant pancreas during follow-up evaluation after pancreatectomy for IPMNs have not been well known. The aim of this study was to investigate the outcomes after resection of IPMNs, especially focusing on the metachronous occurrence of multifocal IPMNs and distinct PDACs. METHODS Medical records of 172 patients who underwent resection of IPMNs were reviewed retrospectively, and the data regarding the occurrence of metachronous IPMNs or PDACs in the remnant pancreas during a mean postoperative follow-up period of 64 months were collected. RESULTS The incidence including synchronous and metachronous multifocal occurrence of IPMNs was 20% (34 of 172), and that of distinct PDACs was 9.9% (17 of 172). Ten metachronous IPMNs developed in the remnant pancreas after a mean time of 23 postoperative months (range, 12-84 mo), and 2 with main duct IPMNs (both were carcinoma in situ) required remnant pancreatectomy. Six distinct PDACs developed in the remnant pancreas after a mean time of 84 postoperative months (range, 12-150 mo). Four of them were found to have a tumor with a size of less than 2 cm, whereas the remaining 2 PDACs were found to be unresectable more than 10 years after resection of IPMNs. CONCLUSIONS Intense long-term follow-up evaluation is necessary for the early detection of metachronous occurrence of distinct PDACs as well as malignant IPMNs after resection of IPMNs.

Modulation of p53 and p73 levels by cyclin G: implication of a negative feedback regulation

Cyclin G is a transcriptional target gene of tumor suppressor p53. Recent studies present evidence that cyclin G may play a central role in the p53-Mdm2 autoregulated module, but the precise function of cyclin G remains elusive. Here, we show a negative effect of cyclin G on the stability of p53 and p73. Cyclin G expression resulted in a dramatic decrease of p53 protein levels in response to DNA damage and abrogated irradiation-mediated G1 arrest along with an increase of S phase in MCF7 cells containing wild-type p53. In p53-null Saos2 cells, cyclin G inhibited p73 induction in response to genotoxic stress and delayed the camptothecin-mediated cell cycle arrest. Cyclin G interacts with p53 as well as p73, and its binding to p53 or p73 presumably mediates downregulation of p53 and p73. We also found that cyclin G-mediated reduction of p53 but not of p73 is Mdm2-dependent. Moreover, inhibition of cyclin G by small interfering RNA (siRNA) caused the accumulation of p53 and p73 protein levels in response to DNA damage. Therefore, our results imply that cyclin G is transcriptionally activated by p53 or p73, and, in turn, cyclin G negatively regulates the stabilization of p53 family proteins through an unknown mechanism different from ubiquitination or transcriptional control.

Induction of hepatocyte growth factor activator gene expression under hypoxia activates the hepatocyte growth factor/c-Met system via hypoxia inducible factor-1 in pancreatic cancer

Hepatocyte growth facor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. Our previous study demonstrated that tumor–stromal interaction under hypoxia augments the aggressive invasive features of pancreatic cancer line PK8 through activated HGF/c‐Met signaling. The present study investigated whether or not hypoxia increases HGFA expression in PK8 cells and promotes the processing of HGF, and leads to c‐Met activation. Moreover, HGFA promoter assays were performed to define whether hypoxia inducible factor‐1 alpha (HIF‐1α) directly activates the HGFA promoter in a hypoxia‐dependent fashion. As a result, hypoxia induced the HGFA mRNA and protein expression in PK8 and the elevation under hypoxia was inhibited by the transfection of HIF‐1α siRNA, thus indicating HIF‐1α‐dependent induction of HGFA. The transfection of siRNA against HGFA to PK8 cells suppressed the conversion to the active HGF, which is secreted from fibroblast MRC5. Furthermore, the phosphorylation of c‐Met and cancer invasion of PK8 cells were decreased by the transfection of HGFA siRNA under hypoxia. Using the luciferase reporter system, HIF‐1α was shown to transactivate the HGFA promoter under hypoxia. These experiments demonstrated for the first time that HGFA is a novel HIF‐1 target gene. Under hypoxia, HGFA might be overexpressed and secreted from pancreatic cancer cells, which contributes to accelerate processing of HGF from fibroblast, resulting in the activation of the c‐Met pathway. HGF/HGFA/c‐Met recruited between cancer‐stromal fibroblasts is activated under hypoxic conditions and therefore might play a central role in the aggressive invasion of pancreatic cancer. (Cancer Sci 2008; 99: 1341–1347)

Intraductal Papillary Mucinous Neoplasms of the Pancreas With Distinct Pancreatic Ductal Adenocarcinomas Are Frequently of Gastric Subtype

Objective:To identify a high-risk group of patients with pancreatic ductal adenocarcinoma (PDAC), independently arising in the pancreas with intraductal papillary mucinous neoplasm (IPMN), using histopathologic subtypes. Background:Pathologic features of IPMN with distinct PDAC, including histopathologic subtypes of IPMN and PDAC phenotypes, have not been well characterized. Mucin expression patterns and the mutational status of GNAS and KRAS are useful to explore the relationship between these 2 lesion types. Methods:Clinicopathologic data of 179 resected IPMNs and 180 resected PDACs without IPMNs as a control group were reviewed. IPMNs were classified into 4 grades (low-grade, intermediate-grade, high-grade dysplasia, and an associated invasive carcinoma) and 4 subtypes (gastric, intestinal, pancreatobiliary, and oncocytic). The expression of MUC1, MUC2, MUC5AC, MUC6, and CDX2 was investigated by immunohistochemistry in IPMNs and PDACs with and without IPMNs. The mutational status of GNAS and KRAS was evaluated by cycle sequencing in PDACs and pre-/coexisting IPMNs. Results:Twenty-six synchronous or metachronous PDACs were identified in 20 patients (11.2%) with IPMNs. Occurrence of concomitant PDACs was more frequently observed in gastric-type IPMNs (18/110, 16.4%) compared with intestinal (1/49, 2.0%), pancreatobiliary (1/17, 5.9%), or oncocytic-type (0/3, 0%) (P = 0.047). Both PDACs with and without IPMNs were frequently positive for MUC1, MUC5AC, and MUC6 expression, as assessed by immunohistochemistry, but were negative for MUC2 and CDX2. The mucin-staining patterns were similar to those of invasive tubular adenocarcinoma arising from gastric-type IPMNs. Mutation of GNAS within codon 201 was not detected in PDACs and gastric-type IPMNs, whereas most of these exhibited KRAS mutations. However, the R201H GNAS mutation was detected in 1 intestinal-type IPMN with distinct PDAC. Conclusions:Mucin expression patterns demonstrate that PDAC without GNAS mutations of an aggressive phenotype frequently arise in the pancreas with benign gastric-type IPMN in the absence of GNAS mutations.