Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.

The identification of cancer biomarkers is critical for target-linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non-neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0 ⁄ 16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0 ⁄ 16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0 ⁄ 16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0 ⁄ 16:1) to lyso-PC(16:0) was higher in CRC, we postulated that lyso-PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC (16: (1) Previous studies have reported that CRC contains increased amounts of phospholipids as well as an altered phospholipid composition of the CRC cell membrane.(2,3) These changes in membrane phospholipid levels can affect cell proliferation, viability and tumor development.(2,4) Moreover, although phosphatidylcholine (PC) is the most dominant phospholipid in both non-neoplastic and cancer tissues,the amount of PC is highly increased in CRC cells.(2) In addition, the changes in membrane potential and the increased PC ⁄ phosphatidylethanolamine (PE) composition rate are related to the grade of CRC malignancy.(3) In higher eukaryotes, PC is synthesized via two pathways: (i) the triple methylation of PE; and (ii) the cytidine diphosphate (CDP)-choline pathway. (6) However, the steady-state composition of PC species is maintained by the remodeling cycle (Lands' cycle).(7) The precise and concerted deacylation by phospholipase A 2 (PLA 2 ) and reacylation by lyso-PC acyltransferase (LPCAT) are required for normal cell functioning. Notably, the elevated expression of LPCAT1 was associated with colon cancer growth. (8) LPCAT1 is a member of the LPCAT family (LPCAT1-4) and shows LPCAT activity, preferentially incorporating palmitate into PC.(8) However, the relationship between PC remodeling and the progression of cancer, as well as the class of LPCAT involved in this process, remains unclear.Direct mass spectrometry (MS) of biological tissue sections using matrix-assisted laser desorption ⁄ ionization (MALDI) can profile many molecules including phospholipid subtypes. (9) Furthermore, this approach can be extended to imaging MS, which can visualize the distribution of biomolecules in the tissue section.(10-13) Because specific antibodies against lipids and macromolecules are often difficult to obtain, MALDI imaging is a suitable option for detecting distinct species of these molecules directly in a tissue section. This technique has already been applied to various human cancers including prostate and gastric cancer. (14,15) Although this emerging analytical technique was initial...

PURPOSE The International Union Against Cancer highlighted tumor budding as a tumor-related prognostic factor. International assessment criteria for tumor budding were recently defined by the 2016 International Tumor Budding Consensus Conference (ITBCC2016). This study aimed to clarify the prognostic and predictive values of tumor budding in a randomized controlled trial evaluating the superiority of adjuvant chemotherapy with oral tegafur-uracil over surgery alone for stage II colon cancer (SACURA trial; ClinicalTrials.gov identifier: NCT00392899 ). PATIENTS AND METHODS Between 2006 and 2010, we enrolled 991 patients from 123 institutions with stage II colon cancer. Tumor budding was diagnosed by central review on the basis of the criteria adopted in the ITBCC2016. We prospectively recorded all clinical and pathologic data, including the budding grade, and performed prognostic analyses after 5 years of completing the patients’ registration. RESULTS Of 991 tumors, 376, 331, and 284 were classified as BD1, BD2, and BD3, respectively; the 5-year relapse-free survival (RFS) rate was 90.9%, 85.1%, and 74.4%, respectively ( P < .001), and ranged widely in T4 tumors (86.6% to 53.3%). The budding grade significantly correlated with recurrence in the liver, lungs, lymph nodes, and peritoneum ( P < .001 to .01). Multivariable analysis revealed that budding and T stage exerted an independent impact on RFS, and on the basis of the Harrell concordance index, these two factors substantially contributed to the improvement of the Cox model for predicting RFS. Both the BD2 and BD3 groups demonstrated greater improvement in the 5-year recurrence rate in the adjuvant chemotherapy group than the surgery-alone group by approximately 5%, but the difference was statistically nonsignificant. CONCLUSION Tumor budding grade on the basis of the ITBCC2016 criteria should be routinely evaluated in pathologic practice and could improve the benefit of adjuvant chemotherapy for stage II colon cancer.

The clinicopathological characteristics relevant to prognosis after surgical treatment of intrahepatic cholangiocarcinoma (ICC) remain unclear. In this study, the clinicopathological features of 19 patients with mass-forming ICC, the most common form of the disease, were reviewed to analyze prognostic determinants. Two or more segmentectomies of the liver with systematic lymphadenectomy were performed in 18 patients. Resection of the extrahepatic bile duct was performed in 14 patients, and reconstruction of the portal vein was accomplished in 5 patients. Stage IVA or IVB tumors were seen in 13 patients, and lymph node (LN) metastasis was present in 14 patients. The estimated 5-year survival rate after surgery for mass-forming ICC was 28%, with median survival time of 18 months. In univariate analysis, five variables were determined to be significantly correlated with poor survival of patients with mass-forming ICC after surgery. These variables include mass-forming ICC with periductal infiltration, perineural invasion, portal vein invasion, presence of intrahepatic metastasis, and two or more LN metastases. Survival rates of 5 patients without LN metastasis and 6 patients with a single LN metastasis were 80% and 33% at 5 years, respectively, while 8 patients with two or more LN metastasis failed to survive beyond 2 years. Multivariate analysis revealed the presence of intrahepatic metastasis to be an independent prognostic factor of poor survival. Hepatectomy with resection of the extrahepatic bile duct and systematic lymphadenectomy yields a good chance for prolonged survival for patients with mass-forming ICC when the lesion is singular and LN metastasis is limited to a regional LN. Because the presence of intrahepatic metastasis was closely related to a poor prognosis in patients with mass-forming ICC, efficacious chemotherapy would be needed to control development of the lesion.