KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30-40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apc(flox/flox); LSL-Kras(G12D) and CDX2P-G22Cre;Apc(flox/flox) mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT-PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin-nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin-NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Oxaliplatin and molecular-targeted drug therapies improved the overall survival in colorectal cancer patients with synchronous peritoneal carcinomatosis undergoing incomplete cytoreductive surgery.

PURPOSE: To estimate the feasibility and limitations of incomplete cytoreductive surgery and modern systemic chemotherapy in patients with synchronous peritoneal carcinomatosis from colorectal cancer and to identify risk factors for death and factors associated with the patient prognosis. METHODS: Sixty-five consecutive patients underwent surgery for synchronous peritoneal carcinomatosis from colorectal cancer at Hiroshima University, Japan between 1992 and 2012. The clinical, histological, and survival data were analyzed for independent risk factors and prognostic factors. The patients were retrospectively stratified into two groups according to the extent of surgery: complete cytoreductive surgery or incomplete cytoreductive surgery. RESULTS: The median survival times in the complete and incomplete cytoreductive surgery groups were 29.8 and 10.0 months, respectively. Receiving systemic chemotherapy alone was an independent risk factor for death in the incomplete cytoreductive surgery group (P < 0.001). Oxaliplatin and molecular-targeted drug (cetuximab or bevacizumab) therapies were also independent prognostic factors (P < 0.001), whereas irinotecan therapy was not a prognostic factor (P = 0.494). CONCLUSION: Oxaliplatin and molecular-targeted drug therapies improved the overall survival in patients undergoing incomplete cytoreductive surgery. Future trials for patients with synchronous peritoneal carcinomatosis from colorectal cancer should be undertaken, with patients stratified according to treatment with complete cytoreductive surgery or incomplete cytoreductive surgery with modern chemotherapy.

Single-incision laparoscopic colectomy using the Gelport system for early colon cancer.

BACKGROUND: Laparoscopic surgery has spread quickly during the past twenty years, and has become one of the important treatments in the field of colorectal surgery. Recently, natural orifice transluminal endoscopic surgery (NOTES) has been studied as the next generation of minimally-invasive surgery, but the feasibility and safety of the NOTES method have not been evaluated. In such a situation, single-incision laparoscopic surgery has attracted interest from surgeons worldwide. However, single-incision laparoscopic colorectal surgery has not yet been standardized. METHODS: From February 2010, single-incision laparoscopic colectomy was performed for 7 patients presenting with early colon cancer. All procedures were performed by two experts with the License of Endoscopic Surgical Skill Qualification System (ESSQS) of Japan Society for Endoscopic Surgery (JSES) in the field of colorectal Surgery. RESULTS: We used the Gelport system (Applied Medical, Rancho Santa Margarita, CA, USA) as the access port and 3 trocars of different sizes (Ethicon, Inc., Cincinnati, OH, USA). Using this technique, we did not experience any difficulties or use any articulated instruments. All of the present 7 patients underwent the single-incision laparoscopic colectomy successfully and had no complications. CONCLUSION: Single-incision laparoscopic surgery using the Gelport was performed safely in the present cases. The use of the Gelport as an access port can address the technical difficulty associated with this new technique.

Helicobacter pylori-induced atrophic gastritis progressing to gastric cancer exhibits sonic hedgehog loss and aberrant CDX2 expression.

BACKGROUND: The loss of sonic hedgehog is an early change that occurs in the mucosa prior to neoplastic transformation and correlates with the type of intestinal metaplasia. Aberrant expression of CDX has also been shown to correlate with the development of intestinal metaplasia. AIM: To examine CDX2 expression in the non-cancerous mucosa of patients with gastric cancer and compared it to CDX2 expression in controls with intestinal metaplasia. METHODS: Sixty patients who had undergone endoscopic mucosal resection for early gastric cancer and 60 gender- and age-matched controls were studied. Two specimens each were obtained from the greater and lesser curves of the corpus and from the greater curve of the antrum. Expression of CDX2 and sonic hedgehog were evaluated by immunostaining. RESULTS: Gastric cancer was associated with a higher frequency of incomplete intestinal metaplasia (OR = 8.3; 95%CI, 3.7-18.9, P < 0.001). CDX2 negatively correlated with sonic hedgehog expression, however, multivariate analysis revealed that CDX2 correlated with the intestinal metaplasia scores. Sonic hedgehog indices were lower and CDX2 staining in the corpus lesser curve was higher in the cancer group than in the controls. Sonic hedgehog indices in the corpus decreased and CDX2 indices in both areas increased in patients in the ascending order of those without intestinal metaplasia, those with complete intestinal metaplasia and those with incomplete intestinal metaplasia (P < 0.001). CONCLUSIONS: Loss of sonic hedgehog expression and aberrant expression of CDX2 correlates with the type of intestinal metaplasia and may play a role in carcinogenesis.

Expression of CDX2 in normal and neoplastic human colon tissue and during differentiation of an in vitro model system.

BACKGROUND: The Cdx genes are expressed in the colorectal epithelium and are frequently downregulated during tumorigenesis. Overexpression of Cdx genes has been shown previously to result in cellular differentiation. AIM: To study expression of CDX2 in normal and neoplastic human colon using a newly isolated monoclonal antibody. To define expression of CDX1 and CDX2 in an in vitro model system of colorectal tumour progression and to ascertain whether these are subject to regulation during differentiation. METHODS: Normal and neoplastic human colon was immunostained for CDX2. CDX1 and CDX2 expression was assayed in cell lines derived from premalignant colonic adenomas by western blotting. Differentiation was induced by sodium butyrate treatment or post confluent growth, and changes in CDX expression compared with carcinoma cell lines with low levels of CDX expression. RESULTS: CDX2 protein displayed no gradient of expression within the colonic crypt. Cell lines derived from adenomas, with high levels of CDX1 and CDX2, showed no regulation of these proteins when induced to differentiate by butyrate or confluency. CDX expression in these cell lines was independent of their APC or Ras status. CDX1 and CDX2 were expressed at very low levels in some carcinoma cell lines and were modestly upregulated on differentiation but were not restored to levels seen in adenoma cells. CONCLUSION: The lack of significant regulation on cellular differentiation and the absence of a detectable gradient in the crypt implies that CDX2 may confer tissue specificity but may not play the previously suggested role in crypt patterning.

Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon.

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

Inhibition of Wnt signaling pathway by a novel axin-binding protein.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.

Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin.

Adenomatous polyposis coli gene product (APC) functions as a tumor suppressor and its mutations in familial adenomatous polyposis and colorectal cancers lead to the accumulation of cytoplasmic beta-catenin. The molecular mechanism by which APC regulates the stability of beta-catenin was investigated. The central region of APC, APC-(1211-2075), has the beta-catenin- and Axin-binding sites and down-regulates beta-catenin. Glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated beta-catenin slightly in the presence of either APC-(1211-2075) or Axin(delta)(beta)(-catenin), in which the beta-catenin-binding site is deleted, and greatly in the presence of both proteins. The enhancement of the GSK-3 beta-dependent phosphorylation of beta-catenin was eliminated by the APC-binding site of Axin. Axin down-regulated beta-catenin in SW480 cells, but not Axin(delta)(beta)(-catenin). In L cells where APC is intact, Axin(delta)(beta)(-catenin) inhibited Wnt-dependent accumulation of beta-catenin but not Axin-(298-832)(delta)(beta)(-catenin) in which the APC- and beta-catenin-binding sites are deleted. These results indicate that the complex formation of APC and Axin enhances the phosphorylation of beta-catenin by GSK-3 beta, leading to the down-regulation of beta-catenin.

Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis.

POB1 has been identified as a RalBP1-binding protein and has the Eps15 homology (EH) domain. The EH domain-containing proteins have been suggested to be involved in clathrin-dependent endocytosis. To clarify the function of POB1, we purified a protein which binds to the EH domain of POB1 from bovine brain cytosol and identified it as Epsin, which is known to bind to the EH domain of Eps15. Epsin has three Asn-Pro-Phe (NPF) motifs in the C-terminal region, which are known to form the core sequence for the binding to the EH domain. The EH domain of POB1 interacted directly with the region containing the NPF motifs of Epsin. Expression of Epsin in CHO-IR cells inhibited internalization of insulin although it affected neither insulin-binding nor autophosphorylation activities of the insulin receptor. Taken together with the observations that Epsin is involved in internalization of the receptors for epidermal growth factor and transferrin, these results suggest that Epsin is a binding partner of POB1 and their binding regulates receptor-mediated endocytosis.

Malignant mesothelioma originating in the hepatic falciform ligament: report of a case.

We report herein what to our knowledge is the first documented case of malignant mesothelioma arising from the falciform ligament. A 65-year-old woman was admitted to our department for the evaluation of an epigastric mass. An abdominal computed tomography scan, magnetic resonance imaging, and celiac angiogram showed a tumor originating in the falciform ligament. The tumor was subsequently resected, and pathological examination confirmed a diagnosis of malignant mesothelioma originating in the falciform ligament. A metastatic liver tumor was detected 3 years after this operation and a partial liver resection was performed. She is currently off therapy, and no clinical or radiologic evidence of disease has been found for 1 year since her second operation.

Tumor localization studies and surgical treatment in patients with insulinoma.

In the present study, we retrospectively reviewed thirteen consecutive patients with insulinomas including 2 reoperations at our department for insulinomas, from the viewpoint of preoperative localization studies, surgery and long term follow-up. The results of the preoperative localization studies proved to be percutaneous transhepatic portal venous sampling (PTPVS) 6/7, angiography 8/15, ultrasonography (US) 6/11, endoscopic ultrasonography (EUS) 4/4, computed tomography (CT) 3/10, and magnetic resonance imaging (MRI) 2/2. The tumor was visualized by intraoperative ultrasonography (IUS) in 6 of 6 patients (100%). Six patients underwent enucleation, 6 patients underwent distal pancreatectomy, 2 patients underwent subtotal (80%) distal pancreatectomy, and one patient a pylorus preserving pancreaticoduodenectomy (PPPD). Two patients, one of whom belonged to the MEN-I group, underwent reoperations because they had multiple adenomas. The development of iatrogenic diabetes occurred in the case of 3 patients. These results suggest that the use of selected preoperative localization studies (PTPVS and probably EUS) may be very helpful for detecting insulinoma, and that IUS is an essential part of the operative exploration for insulinoma. Our data may further indicate the need for an aggressive surgical procedure in the case of multiple adenoma or insulinoma in MEN-I.

Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral.

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.

Protective effect of monoclonal antibodies to adhesion molecules on rat liver ischemia-reperfusion injury.

BACKGROUND: The source of reactive oxygen species in the liver remains to be elucidated. The present study was undertaken to determine whether polymorphonuclear neutrophils (PMNs) can contribute to hepatic ischemia-reperfusion injury, and pretreatment with monoclonal antibodies (mAbs) to intercellular adhesion molecule-1 (ICAM-1), lymphocyte function associated antigen-1 (LFA-1), and CD 18 could improve energy metabolism and prolong the viability of the organ. METHODS: Male Wistar rats were used. Rat liver ischemia was induced by clamping blood vessels supplying median and left lateral hepatic lobes. Monoclonal antibodies to ICAM-1, LFA-1, or CD18 were injected intravenously 5 minutes before inducing ischemia. To determine the effect of mAbs on the survival rate, total hepatic ischemia was induced by clamping the hepatic artery, portal vein, and bile duct after making a portafemoral shunt. RESULTS: Although ischemia of the liver for 90 minutes did not permit survival of the animals, pretreatment with mAbs to ICAM-1 plus LFA-1 increased the survival rate to 57%. Pretreatment with mAb to ICAM-1 failed to increase the survival rate. The number of PMNs in the liver increased continually up to 24 hours after reperfusion after 90 minutes of ischemia, and the expression of ICAM-1 was enhanced 4 hours after reperfusion. This is accompanied by a low recovery of hepatic adenosine triphosphate and, on the contrary, a marked increase in lipid-peroxide in the reperfused liver. Pretreatment with mAbs suppressed the infiltration of PMNs and the elevation of lipid peroxide and enhanced the recovery of hepatic adenosine triphosphate 6, 12, or 24 hours after reperfusion. Pretreatment with mAbs also prevented the rise in serum alanine aminotransferase level after reperfusion. CONCLUSIONS: These results suggest that PMNs contribute to ischemia-reperfusion injury in the liver 4 hours and more after reperfusion, and pretreatment with mAbs to adhesion molecules is useful for the prevention of ischemic liver cell injury.

Synergistic activation of c-fos promoter activity by Raf and Ral GDP dissociation stimulator.

Ral, a member of small GTP-binding protein (G protein) superfamily, has been suggested to act downstream of Ras, since Ral GDP dissociation stimulator (RalGDS) has been found to be an effector protein of Ras. In this study, we examined the effects of RalGDS and Ral on gene expression using c-fos promoter linked to the luciferase reporter gene (c-fos-luciferase). RalGDS interacted with RasG12V/E37G (in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf in COS cells. RafCAAX is an active Raf kinase targeted to the plasma membranes by virtue of the addition of a C-terminal localization signal from K-Ras. Transfection of either RalGDS or RafCAAX into NIH3T3 cells slightly stimulated c-fos-luciferase expression and cotransfection of both proteins greatly enhanced the expression. RalGDS and an activated Rac (RacG12V) did not act synergistically to stimulate c-fos-luciferase expression. Transfection of an activated Ral (RalG23V) stimulated c-fos-luciferase expression. Furthermore, cotransfection of RalG23V and an activated Ras (RasG12V) enhanced RasG12V-dependent c-fos-luciferase expression. However, RalG23V did not synergize with RafCAAX, RacG12V or RalGDS to stimulate the expression. These results show that RalGDS and Ral regulate c-fos promoter activity and suggest that RalGDS may activate c-fos promoter synergistically with the signal from Raf by transmitting the signal to a target other than Ral.

Post-translational modifications of Ras and Ral are important for the action of Ral GDP dissociation stimulator.

Ral GDP dissociation stimulator (RalGDS) is a GDP/GTP exchange protein of Ral and a new effector protein of Ras. Therefore, there may be a new signaling pathway from Ras to Ral. In this paper, we examined the roles of the post-translational modifications of Ras and Ral on this new signal transduction pathway. The post-translationally modified form of Ras bound to RalGDS more effectively than the unmodified form. The modification of Ras was required to regulate the distribution of RalGDS between the cytosol and membrane fractions in COS cells. The post-translational modification of Ral enhanced the activities of RalGDS to stimulate the dissociation of GDP from and the binding of GTP to Ral. Furthermore, the modified form of Ral bound to Ral-binding protein 1 (RalBP1), a putative effector protein of Ral, more effectively than the unmodified form. Taken together with the observations that Ras and Ral are localized to the membranes, these results suggest that the post-translational modifications of Ras and Ral play a role for transmitting the signal effectively on the membranes in the signal transduction pathway of Ras/RalGDS/Ral/RalBP1.

Ras-interacting domain of Ral GDP dissociation stimulator like (RGL) reverses v-Ras-induced transformation and Raf-1 activation in NIH3T3 cells.

Ral GDP dissociation stimulator (RalGDS) and RalGDS like (RGL) are putative effector proteins of Ras and contain the Ras-interacting domain (RID) at their C-terminal regions. v-Ras is known to activate c-fos promoter/enhancer and Raf-1 and to transform NIH3T3 cells. It is also known that v-Raf activates c-fos promoter/enhancer and transforms NIH3T3 cells. In this study, we examined the effect of RID on the phenotype of the cells transformed by v-Ras and v-Raf. Overexpression of RID greatly reduced cell growth in low serum, colony-forming activity in soft agar, c-fos promoter/enhancer activity, and Raf-1 activity of v-Ras-transformed cells. However, overexpression of RID did not affect the phenotype of v-Raf-transformed cells. These results clearly indicate that RID of RGL specifically binds to Ras in mammalian cells, that it blocks the signal from Ras to Raf-1, and that it reverses v-Ras-induced malignant phenotype. It has been reported that Ras-binding domains of Raf-1 and neurofibromatosis type 1 (NF1) reverse v-Ras-induced malignant phenotype. Since there is no homology in primary structures of RGL, Raf-1, and NF1, there may be a similarity of secondary or tertiary structure among RID of RGL and Ras-binding domains of Raf-1 and NF1, and the structure might be useful for developing a potential medicine for human cancers caused by Ras.

Bacteriological investigation of bile in patients with cholelithiasis.

The microflora in bile from the gallbladder and common bile duct was investigated in 303 patients who underwent surgery for cholelithiasis. The purpose of this study was to identify current bacteria and bacterial casts in the biliary tract and also to analyze the relationship between bactericholia at the time of operation and postoperative infection. Bile cultures were positive in 38% of all patients, although a higher incidence of positive bile cultures occurred in patients over 70 years of age (77%), those with common duct stones (83%), those with pigment stones (65%), and those who underwent gastrectomy (71%). The predominant organisms were Escherichia coli (22%), Klebsiella (18%), and Enterococcus (15%). Obligate anaerobes were less frequently seen (4%), being found only in patients with pigment stones and always mixed with aerobes. Four patients developed postoperative infections (1.3%) which were all caused by biliary bacteria. The following two factors may contribute to this low incidence of postoperative infections: our policy of operating electively whenever possible, and the prophylactic use of antibiotics to which the organisms cultured from bile are sensitive.