Koji Fukase

GCF2/LRRFIP1 promotes colorectal cancer metastasis and liver invasion through integrin-dependent RhoA activation

The precise relationship between GCF2 expression and carcinogenesis has not yet been established. To clarify the metastatic potential of GCF2 in colorectal cancer, HT-29 cells stably suppressing GCF2 expression were injected into the spleens of severe combined immunodeficient (SCID) mice. GCF2 suppression reduced the number of metastatic foci in the liver and reduced fibronectin-induced cell adhesion, migration, and invasion. Downstream from the integrin signaling pathways, GCF2 regulates RhoA interaction with the RGS domain of Leukemia associated RhoGEF (LARG). Altogether, our results suggest that GCF2 plays an important role in colorectal cancer metastasis by regulating RhoA-induced cell adhesion, migration, and invasion.

Bile acids repress E‐cadherin through the induction of Snail and increase cancer invasiveness in human hepatobiliary carcinoma

Although some kinds of bile acids have been implicated in colorectal cancer development, the mechanism of cancer progression remains unexplored in hepatobiliary cancer. From our personal results using complementary DNA microarray, we found that chenodeoxycholic acid (CDCA) induced Snail expression in human carcinoma cell lines derived from hepatocellular carcinoma and cholangiocarcinoma. Snail expression plays an important role in the regulation of E‐cadherin and in the acquisition of invasive potential in many types of human cancers including hepatocellular carcinoma. We found that CDCA and lithocholic acid (LCA) induced Snail expression in a concentration‐dependent manner and down‐regulated E‐cadherin expression in hepatocellular carcinoma and cholangiocarcinoma cell lines. Moreover, Snail short interference RNA (siRNA) treatment reduced the down‐regulation of E‐cadherin by CDCA or LCA. Luciferase analysis demonstrated that the promoter region from –111 to –24 relative to the transcriptional start site was necessary for this induction and, at least in part, nuclear factor Y (NF‐Y) and stimulating protein 1 (Sp1) might be an inducer of Snail expression in response to bile acids. In addition, using an in vitro wound healing assay and invasion assay, we observed that CDCA and LCA induced cell migration and invasion. These results suggest that bile acids repress E‐cadherin through the induction of transcription factor Snail and increase cancer invasiveness in human hepatocellular carcinoma and cholangiocarcinoma. Inhibition of this bile acid‐stimulated pathway may prove useful as an adjuvant in the therapy of hepatocellular carcinoma. (Cancer Sci 2008; 99: 1785–1792)

Silencing of LRRFIP1 reverses the epithelial–mesenchymal transition via inhibition of the Wnt/β-catenin signaling pathway

The canonical Wnt/β-catenin signaling pathway has been shown to promote the epithelial-mesenchymal transition (EMT), which is a crucial process in multiple embryonic developmental processes and the progression of carcinomas. We recently provided evidence that leucine-rich repeat flightless-1-interacting protein 1 (LRRFIP1) promotes cancer metastasis and invasion. In the present study, we identified the signaling elements targeted by LRRFIP1 for promotion of the EMT in pancreatic and lung cancer. LRRFIP1 silencing reversed the EMT, as shown by increased expression of E-cadherin (an epithelial marker) and decreased expression of vimentin (a mesenchymal marker). Silencing of LRRFIP1 up-regulated phosphorylation of β-catenin and decreased its nuclear localization by targeting the β-catenin destruction complex. The expression of β-catenin and E-cadherin in the plasma membrane fraction was increased in LRRFIP1 silenced cancer cells, and the migration and invasion capabilities were strongly inhibited. In addition, this protein was highly expressed at the invasion front of malignant tissue collected from pancreatic cancer patients. Consequently, our data strongly suggested that LRRFIP1 played an important role in the invasion of carcinoma cells. Our data provide experimental evidence that LRRFIP1 is an attractive candidate for targeted therapy in human cancers.

Application of Quantitative Targeted Absolute Proteomics to Profile Protein Expression Changes of Hepatic Transporters and Metabolizing Enzymes During Cholic Acid-Promoted Liver Regeneration

Preoperative administration of cholic acid (CA) may be an option to increase the liver volume before elective liver resection surgery, so it is important to understand its effects on liver functionality for drug transport and metabolism. The purpose of this study is to clarify the absolute protein expression dynamics of transporters and metabolizing enzymes in the liver of mice fed with CA-containing diet for 5 days (CA1) and mice fed with CA-containing diet for 5 days followed by diet without CA for 7 days (CA2), in comparison with non-CA-fed control mice. The CA1 group showed the increased liver weight, cell proliferation index, and oxidative stress, but no increase in apoptosis. Quantitative targeted absolute proteomics revealed (1) decreases in basolateral bile acid transporters Na+-taurocholate cotransporting polypeptide, anion transporting polypeptide (oatp) 1a1, and oatp1b2, bile acid synthesis-related enzymes cyp7a1 and cyp8b1, and drug transporters breast cancer resistance protein, multidrug resistance-associated protein 6, ent1, and oatp2b1; and (2) increases in glutathione biosynthetic enzymes and drug-metabolizing enzyme cyp3a11. Liver concentrations of reduced and oxidized glutathione were both increased. In the CA2 group, the increased liver weight was maintained, whereas the biochemical features and protein profiles were restored to the non-CA-fed control levels. These findings suggest that CA administration alters liver functionality per body during liver regeneration and restoration.

Lymph nodes metastasis is a risk factor for bone metastasis from extrahepatic cholangiocarcinoma.

BACKGROUND/AIMS The rate and site of bone metastasis from cholangiocarcinoma as well as the prognosis are unclear. Therefore, we intend to make a comparative review of the background to bone metastasis, examine a high-risk group for bone metastasis and use the data towards the improvement in quality of life. METHODOLOGY We studied 200 cases of cholangiocarcinoma resected in our division from January 2003 to April 2010. RESULTS Bone metastasis was confirmed in four cases (2.0%). The survival period after the diagnosis of bone metastasis ranged from 2.9 months to 21.6 months and the average was 6.7 months. We studied histopathological findings of bone metastasis, lymph node metastasis, lymphatic invasion, blood vessel invasion and perineural invasion (ly, v and pn) and found that all of four bone metastasis cases were positive for lymph node metastasis which was a statistically significant factor affecting bone metastasis. CONCLUSIONS Since the number of cases we studied is small, it is difficult to determine whether lymph node metastasis is a risk factor for bone metastasis; however, we think it is necessary to take the probability of bone metastasis into consideration when we provide medical care to patients positive for lymph node metastasis.

Surgical Outcome of Extra-hepatic Cholangiocarcinoma

Extrahepatic cholangiocarcinoma is divided into two compartments, namely perihilar cholangiocarcinoma and distal cholangiocarcinoma. Hemihepatectomy and caudate lobectomy with resection of extrahepatic bile duct is usually performed for patients with perihilar cholangiocarcinoma, whereas pancreaticoduodenectomy is performed for distal cholangiocarcinoma. As pancreaticoduodenectomy has been a well-established and more commonly performed operation, distal cholangiocarcinoma was thought to have better outcomes as compared to perihilar cholangiocarcinoma. However, both cholangiocarcinomas originate from the bile duct epithelium and it is not clear if the malignant potential of perihilar cholangiocarcinoma and distal cholangiocarcinoma are different or not. Therefore, we analyzed 162 perihilar and 98 distal cholangiocarcinoma in our institution from 2002 to 2011, to clarify the actual overall survival, disease-free survival, and prognostic factors. The actual 5-year overall survivals of perihilar and distal cholangiocarcinoma were 39.8 % and 45.2%, respectively, and actual 5-year disease-free survivals were 29.6% and 38.4%, respectively. There are no significant differences in overall survival and disease-free survival between perihilar and distal cholangiocarcinoma. In both cholangiocarcinomas groups independent prognostic factors were distant metastasis, lymph node metastases, perineural invasion, and lack of R0 resection. In conclusion, the oncological outcomes of perihilar and distal cholangiocarcinoma are similar poor long-term outcome for both groups. Effective neoadjuvant and adjuvant therapies must be established to improve patients’ prognosis. Key word: distal cholangiocarcinoma, perihilar cholangiocarcinoma, biliary tract cancer, prognostic factor, overall survival

FBXW7 modulates malignant potential and cisplatin-induced apoptosis in cholangiocarcinoma via NOTCH1 and MCL1

The ubiquitin ligase F‐box and WD repeat domain‐containing 7 (FBXW7) is responsible for degrading diverse oncoproteins and is considered a tumor suppressor in many human cancers. Inhibiting FBXW7 enhances the malignant potential of several cancers. In this study, we aimed to investigate the role of FBXW7 in cholangiocarcinoma. We found that FBXW7 expression was associated with clinicopathological outcomes in cholangiocarcinoma patients. Both disease‐free and overall survival were significantly worse in the low‐FBXW7 group than in the high‐FBXW7 group (P = .001 and P < .001, respectively). Multivariate analysis with the Cox proportional hazards model indicated that FBXW7 was the most important independent prognostic factor for disease‐free (P = .006) and overall (P = .0004) survival. We also showed that the two FBXW7 substrates, NOTCH1 and myeloid cell leukemia sequence 1 (MCL1), regulate cholangiocarcinoma progression. Depletion of FBXW7 resulted in NOTCH1 accumulation and increased cholangiocarcinoma cell migration and self‐renewal. Interestingly, when cells were stimulated with cis‐diamminedichloridoplatinum(II) (cisplatin), FBXW7 suppression induced MCL1 upregulation, which reduced the sensitivity of cholangiocarcinoma cells to apoptosis, indicating that FBXW7‐mediated ubiquitylation is context‐dependent. These results indicate that FBXW7 modulates the malignant potential of cholangiocarcinoma through independent regulation of NOTCH1 and MCL1.

MK2461, a Multitargeted Kinase Inhibitor, Suppresses the Progression of Pancreatic Cancer by Disrupting the Interaction Between Pancreatic Cancer Cells and Stellate Cells

Objectives Platelet-derived growth factor receptor beta (PDGFR&bgr;) and hepatocyte growth factor receptor (MET) expressed on pancreatic stellate cells (PSCs) are suggested as important components modulating the interactions between pancreatic cancer cells (PCCs) and PSCs. The objective of this study is to clarify the effect of MK2461, a multikinase inhibitor targeting MET and PDGFR&bgr;, on the interaction between PCCs and PSCs. Methods In this study, we profiled the expression of receptor tyrosine kinases (including PDGFR&bgr; and MET) in pancreatic cancer with quantitative targeted absolute proteomics using liquid chromatography tandem mass spectrometry. In addition, the effect of MK2461 on PCC-PSC interaction was investigated using PSCs prepared from pancreatic cancer tissues. Results In PSCs, PDGFR&bgr; and MET were upregulated compared with other receptor tyrosine kinases. Conditioned medium from PSCs promoted the proliferation of PCCs, and vice versa. Moreover, MK2461 suppressed the effects of conditioned medium on PCCs and PSCs. Finally, MK2461 significantly inhibited tumor growth in mice coinjected with PCCs and PSCs. Conclusions The PDGFR&bgr; and MET may play a critical role in the interaction between PCCs and PSCs, which was modulated by MK2461. Therefore, MK2461 may have therapeutic potential in the treatment of pancreatic cancer.

The comparative anatomy of the folds, fossae, and adhesions around the duodenojejunal flexure in mammals

BACKGROUND Anatomical knowledge of the duodenojejunal flexure is necessary for abdominal surgeries, and also important for physiologic studies about the duodenum. But little is known about the anatomy of this region in mammals. Here, we examined comparative anatomy to understand the anatomical formation of the duodenojejunal flexure in mammals. MATERIALS AND METHODS The areas around the duonenojejunal flexure were ob-served in mouse, rat, dog, pig, and human, and the anatomical structures around the duodenojejunal junction in the animals were compared with those in human. RESULTS The superior and inferior duodenal folds, and the superior and inferior duodenal fossae were identified in all examined humans. In pig, the structures were not clearly identified because the duodenum strongly adhered to the retroperitoneum and to the mesocolon. In mouse, rat, and dog, only the plica duodenocolica, which is regarded as the animal counterpart of the superior duo-denal fold in human, was identified, and other folds or fossae were not observed, probably because the duodenum was not fixed to the parietal peritoneum in those animals. Transection of the plica duodenocolica could return the normally rotated intestine back to the state of non-rotation in rat. CONCLUSIONS This study showed the anatomical similarities and dissimilarities of the duodenojejunal flexure among the mammals. Anatomical knowledge of the area is useful for duodenal and pancreatic surgeries, and for animal studies about the duodenum. (Folia Morphol 2018; 77, 2: 286-292).

Abstract 404: MK2461 suppress progression of pancreatic cancer disrupting interaction between pancreatic cancer cells and stellate cells

Pancreatic cancer is characterized excessive desmoplasia, which occupies 80% of pancreatic cancer tissue and mainly consists of pancreatic stellate cells (PSCs). Desmoplasia has been shown to play an important role in the progression, invasion, and metastasis of pancreatic cancer and has been implicated in the development of resistance to chemotherapy and radiotherapy. Growth factors are one of the components modulating the interactions between pancreatic cancer cells (PCCs) and PSCs. Therefore, blocking growth factor signaling could suppress cancer progression. In this study, we profiled the expression of receptor tyrosine kinases (RTKs) with quantitative targeted absolute proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) in PCCs and PSCs. In PCCs, epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) levels were elevated compared with those in PSCs. In PSCs, platelet-derived growth factor receptor beta (PDGFRβ) and MET were upregulated compared with other RTKs. Conditioned medium from PSCs promoted the proliferation of PCCs, and vice versa. In addition, MK2461, a multikinase inhibitor targeting MET and PDGFRβ, suppressed the effects of conditioned medium on PCCs and PSCs. In addition, MK2461 significantly inhibited tumor growth in mice xenograft model. In conclusion, PDGFRβ and MET may play a critical role in the interaction between PCCs and PSCs. Moreover, MK2461 may have therapeutic potential in the treatment of pancreatic cancer by targeting the interaction between PCCs and PSCs. Citation Format: Koetsu Inoue, Hideo Ohtsuka, Fuyuhiko Motoi, Daisuke Douchi, Shuhei Kawasaki, Kei Kawaguchi, Kunihiro Masuda, Koji Fukase, Shinobu Ohnuma, Takeshi Naitoh, Yu Katayose, Shinichi Egawa, Michiaki Unno. MK2461 suppress progression of pancreatic cancer disrupting interaction between pancreatic cancer cells and stellate 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 404. doi:10.1158/1538-7445.AM2015-404