Katsunobu Taki

GNAS R201H and Kras G12D cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm

Intraductal papillary mucinous neoplasm (IPMN), the most common pancreatic cystic neoplasm, is known to progress to invasive ductal adenocarcinoma. IPMNs commonly harbor activating somatic mutations in GNAS and KRAS, primarily GNASR201H and KRASG12D. GNAS encodes the stimulatory G-protein α subunit (Gsα) that mediates a stimulatory signal to adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), subsequently activating cAMP-dependent protein kinase A. The GNASR201H mutation results in constitutive activation of Gsα. To study the potential role of GNAS in pancreatic tumorigenesis in vivo, we generated lines of transgenic mice in which the transgene consisted of Lox-STOP-Lox (LSL)-GNASR201H under the control of the CAG promoter (Tg(CAG-LSL-GNAS)). These mice were crossed with pancreatic transcription factor 1a (Ptf1a)-Cre mice (Ptf1aCre/+), generating Tg(CAG-LSL-GNAS);Ptf1aCre/+ mice. This mouse line showed elevated cAMP levels, small dilated tubular complex formation, loss of acinar cells and fibrosis in the pancreas; however, no macroscopic tumorigenesis was apparent by 2 months of age. We then crossed Tg(CAG-LSL-GNAS);Ptf1aCre/+ mice with LSL-KrasG12D mice, generating Tg(CAG-LSL-GNAS);LSL-KrasG12D;Ptf1aCre/+ mice. We used these mice to investigate a possible cooperative effect of GNASR201H and KrasG12D in pancreatic tumorigenesis. Within 5 weeks, Tg(CAG-LSL-GNAS);LSL-KrasG12D;Ptf1aCre/+ mice developed a cystic tumor consisting of marked dilated ducts lined with papillary dysplastic epithelia in the pancreas, which closely mimicked the human IPMN. Our data strongly suggest that activating mutations in GNAS and Kras cooperatively promote murine pancreatic tumorigenesis, which recapitulates IPMN. Our mouse model may serve as a unique in vivo platform to find biomarkers and effective drugs for diseases associated with GNAS mutations.

SPINK1 status in colorectal cancer, impact on proliferation, and role in colitis-associated cancer

Colorectal cancer is a major cause of deaths due to cancer; therefore, research into its etiology is urgently needed. Although it is clear that chronic inflammation is a risk factor for colorectal cancer, the details remain uncertain. Serine protease inhibitor, Kazal type 1 (SPINK1) is mainly produced in pancreatic acinar cells. However, SPINK1 is expressed in various cancers and in inflammatory states, such as colon cancer and inflammatory bowel disease. There are structural similarities between SPINK1 and epidermal growth factor (EGF). Hence, it was hypothesized that SPINK1 functions as a growth factor for tissue repair in inflammatory states, and if prolonged, acts as a promoter for cell proliferation in cancerous tissues. Here, immunohistochemical staining for SPINK1 was observed in a high percentage of colorectal cancer patient specimens and SPINK1 induced proliferation of human colon cancer cell lines. To clarify its role in colon cancer in vivo, a mouse model exposed to the colon carcinogen azoxymethane and nongenotoxic carcinogen dextran sodium sulfate revealed that Spink3 (mouse homolog of SPINK1) is overexpressed in cancerous tissues. In Spink3 heterozygous mice, tumor multiplicity and tumor volume were significantly decreased compared with wild-type mice. These results suggest that SPINK1/Spink3 stimulates the proliferation of colon cancer cells and is involved in colorectal cancer progression. Implications: Evidence suggests that SPINK1 is an important growth factor that connects chronic inflammation and cancer. Mol Cancer Res; 13(7); 1130–8. ©2015 AACR.

Intraoperative blood loss is not a predictor of prognosis for pancreatic cancer

PurposeThe relationship between intraoperative blood loss (IBL) and prognosis has been reported for some types of cancer, but not for pancreatic cancer, which has one of the highest mortality rates of any cancer. We conducted this study to analyze the relationship between IBL and clinical outcome for patients undergoing radical surgery for pancreatic cancer.MethodsThe subjects of this study were 144 patients who underwent curative pancreatectomy for invasive pancreatic cancer between 2002 and 2014. Clinicopathological characteristics were recorded and prognostic factors were identified by univariate and multivariate analyses.ResultsLarge IBL was significantly associated with advanced tumor stage, a long operation time, a large tumor, portal vein resection, and blood transfusion. According to univariate analysis, IBL was also significantly associated with overall survival (OS) and relapse-free survival (RFS); however, it was not an independent prognostic factor for OS and RFS in multivariate analysis. According to multivariate analysis, lymph node metastasis and R-status were independent prognostic factors for OS and RFS. A subgroup analysis of patients who received no blood transfusion showed similar results.ConclusionMinimizing IBL is very important; however, the present study found that positive lymph node metastasis and R-status were stronger independent prognostic factors for pancreatic cancer.

Heterogeneity of KRAS Mutations in Pancreatic Ductal Adenocarcinoma

Objectives Activating Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are the most common and frequent changes observed in pancreatic cancer. This study aimed to determine the frequency and extent of intratumoral and metastatic lymph node KRAS mutation heterogeneity of resected pancreatic ductal adenocarcinoma. Methods Tumor tissues macrodissected from tumor centers, invasion fronts (n = 97), and lymph nodes (n = 11) were subjected to DNA extraction and mutation analysis of KRAS codons 12 and 13 by pyrosequencing. Results Activating mutations in codon 12 of KRAS were detected in 90 (92.8%) tumor centers. No mutations were detected in KRAS codon 13 in any patient. After a comparison of tumor centers and invasion fronts, intratumoral heterogeneity of KRAS was observed only in 4 (4.1%) cases. Additional invasion front tumor analysis revealed the same mutation status consistent with each tumor center. No heterogeneity was observed between primary tumors and metastatic lymph nodes. Conclusions Intratumoral heterogeneity of the KRAS mutational status is rare in pancreatic ductal adenocarcinoma. In addition, no KRAS heterogeneity between primary tumors and metastatic lymph nodes was detected in this study. This finding is consistent with the hypothesis that oncogenic activation of KRAS is the first driver mutation in pancreatic cancer.

Preoperative platelet-to-lymphocyte ratio can predict recurrence beyond the Milan criteria after hepatectomy for patients with hepatocellular carcinoma.

Postoperative recurrence beyond the Milan criteria is a poor prognostic factor for patients with hepatocellular carcinoma (HCC) treated with various therapies. We investigated the most useful inflammation‐based prognostic score for predicting recurrence beyond the Milan criteria after initial liver resection.

Giant liposarcoma of the posterior mediastinum and retroperitoneum.

A 39-year-old Japanese man has been complaining of chest pain for 3 months and a CT revealed a huge tumour in the posterior mediastinum and retroperitoneum. The mediastinal tumour was continuous with the retroperitoneal one through the oesophageal hiatus and the lower oesophagus was completely surrounded by the tumour. MRI suggested lipogenic tumour. Resection of the tumour with oesophagectomy was performed through bilateral thoracotomy and laparotomy. Pathological examination revealed well-differentiated liposarcoma which was completely resected. The patient has been alive without recurrence for 14 months after surgery.

Abstract 4195: GNASR201H and KrasG12D cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm

Intraductal papillary mucinous neoplasm (IPMN) is the most common pancreatic cystic neoplasm, which is a precursor of the invasive ductal adenocarcinoma. IPMNs recurrently harbor activating somatic mutations in GNAS and KRAS, mostly GNASR201H and KRASG12D. GNAS encodes the stimulatory G-protein α subunit (Gsα) that mediates a stimulatory signal to adenylyl cyclase to produce cyclic AMP (cAMP), a second messenger activating cAMP dependent protein kinase (PKA). The GNASR201H mutation is known to result in constitutive activation of Gsα and its downstream signaling molecules, which induces expression of various genes including mucin genes. To study a role of GNAS in pancreatic tumourigenesis in vivo, we generated transgenic mice lines of which transgene is consisted of Lox-STOP-Lox (LSL)- GNASR201H under the control of the CAG promoter (Tg(CAG-LSL-GNAS)). The transgene contains genetic elements that inhibit transcription and translation of GNASR201H flanked by functional LoxP sites. These mice lines were crossed to pancreatic transcription factor 1 a (Ptf1a)-cre mice, and generated Tg(CAG-LSL-GNAS);Ptf1a-cre mice. This mouse line showed elevated cAMP levels and a compensatory upregulation of PKA in the pancreas. GNASR201H alone was not sufficient to induce tumourigenesis by 2 months of age, but small dilated tubular complex formation, loss of acinar cells, and fibrosis, which could be a very early phenotype of IPMN. To know a cooperative effect of GNASR201H and KRASG12D in pancreatic tumorigenesis, we crossed Tg(CAG-LSL-GNAS);Ptf1a-cre mice with LSL-KrasG12D mice. Tg(CAG-LSL-GNAS);LSL-KrasG12D;Ptf1a-cre mice developed multiple cysts consisting of marked dilated ducts lined with papillary dysplastic epithelia in the pancreas within 5 weeks, which recapitulated human IPMNs. Our data strongly suggest that mutated GNAS cooperating with mutated KRAS may sufficiently cause to develop IPMN. Citation Format: Toru Furukawa, Etsuko Tanji, Masaki Ohmuraya, Katsunobu Taki, Kimi Araki. GNASR201H and KrasG12D cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4195. doi:10.1158/1538-7445.AM2015-4195

Abstract 3125: miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells

Background: Hepatocellular carcinoma is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. Despite recent advance in cancer treatment including the development of molecular target drugs, the prognosis in patients with hepatocellular carcinoma (HCC) still remains poor. The inactivation of the Hippo-pathway lead to TAZ/YAP overexpression, and is associated with worse prognostic outcomes in various cancers including hepatocellular carcinoma (HCC). While there are several reports of miR targeting for YAP, miR targeting for TAZ remains unclear. The aim of this study is to identify the miR targeting TAZ expression in HCC. Methods: miR expression was analyzed using the Human miFinder 384HC miScript miR PCR Array and was compared between low and high TAZ expression cell lines. Then, we extracted miR-9-3p as a tumor-suppressor miR targeting TAZ using miRBase. We examined the functional role of miR-9-3p targeting TAZ using miR-9-3p mimic and inhibitor in HCC cell lines (HLF and HuH1 with high and low TAZ expressions, respectively). Results: In HCC cell lines and HCC clinical samples, there was the inverse correlation between miR-9-3p and TAZ expressions by real-time PCR. By western blotting, TAZ expression was induced by treatment of miR-9-3p inhibitor and was downregulated by treatment of miR-9-3p mimic. TAZ nuclear localization was indeed inhibited with a treatment with miR-9-3p mimics in HLF cells, and was inversely accelerated with a treatment of miR-9-3p inhibitors in HuH1 cells. Treatment of miR-9-3p mimic inhibited cell proliferative ability by downregulating the phosphorylation of Erk1/2 and Akt signals in HLF. Inversely, treatment of miR-9-3p inhibitor accelerated cell growth compared to control in HuH1. On the other hand, cell invasiveness was not affected by miR-9-3p. Conclusions: miR-9-3p was identified as the tumor-suppressor miR targeting TAZ expression in HCC cells. miR-9-3p played a crucial role in cell proliferation, but not in invasion, via Akt and Erk1/2 signals in HCC cells. Citation Format: Takaaki Higashi, Hiromitsu Hayashi, Hideaki Takeyama, Takayoshi Kaida, Kota Arima, Katsunobu Taki, Hirohisa Okabe, Hidetoshi Nitta, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. miR-9-3p plays a tumor-suppressor role by targeting TAZ (WWTR1) in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2015-3125

Clinical significance of half-lives of the tumor markers alpha-fetoprotein and des-γ-carboxy prothrombin after hepatectomy for hepatocellular carcinoma

The prognostic significance of the half‐lives (HLs) of α‐fetoprotein (AFP) and des‐γ‐carboxy prothrombin (DCP) in patients undergoing hepatectomy for hepatocellular carcinoma (HCC) is unclear. We evaluated the HLs of AFP and DCP in a cohort of such patients.

Degenerative hepatic hemangioma mimicking biliary cystadenocarcinoma

A small subset (3% to 5%) of liver cystic lesions represents biliary cystic tumor. Biliary cystic tumor arises from the biliary epithelium and can be classified as biliary cystadenoma and cystadenocarcinoma. Liver resection is the only curative treatment; however, long-term outcomes after resection of biliary cystadenocarcinoma are not always satisfactory. A 74-year-old man was diagnosed with a simple liver cyst 10 years earlier at the affiliated hospital. He was referred there again for the treatment of an enlarged cystic liver lesion with mural nodules. He had no specific symptoms and no obvious abnormality on physical examination. The levels of CEA (7.9 ng/ml) and CA19-9 (48.7U/ml) were slightly increased. Contrast-enhanced CT revealed a well-circumscribed unilocular low-attenuation mass with gradually enhancing mural nodular components (Fig. 1a). Heavily T2-weighted MR image showed a cystic lesion with a fluid-fluid level (Fig. 1b). The inferior fluid layer is hypointense relative to the superior fluid layer. PET/CT showed a slight increase in FDG-activity accordant with a mural nodule in cystic tumor (Fig. 1c). The patient was diagnosed with a typical biliary cystadenocarcinoma of the liver and received extended right hepatectomy. The macroscopic specimen showed a cystic tumor with a mural nodule containing denatured liquid components (Fig. 1d). Pathologically, the cystic tumor was diagnosed as a degenerative hemangioma with an encapsulated hematoma. The mural nodule consisted of remnant hemangioma, outgrowth of dilated blood vessels, intratumoral hemorrhage, and deposition of fibrin and hemosiderin. The patient was discharged on postoperative day 12 with no serious complications and is alive without any symptom and recurrence for 1 year. This is a case of a degenerative hepatic hemangioma mimicking biliary cystadenocarcinoma. A hepatic cavernous