To cut or not to cut? A prospective randomized controlled trial on short-term outcomes of the uncut Roux-en-Y reconstruction for gastric cancer.

BACKGROUND: Roux-en-Y (R-Y) anastomoses have been widely used in distal gastrectomy, while the incidence of Roux stasis syndrome remains common. Uncut R-Y anastomosis maintains the neuromuscular continuity, thus avoiding the ectopic pacemaker of the Roux limb and reducing the occurrence of Roux stasis. However, retrospective studies of Uncut R-Y anastomosis remain scarce and randomized controlled trials have not been reported. METHODS: We conducted a randomized controlled trial to compare the surgical safety, nutritional status, and postoperative quality of life (QOL) between uncut and classic Roux-en-Y (R-Y) reconstruction patients. Patients with Stage I gastric cancer were randomly enrolled and underwent laparoscopic distal gastrectomy followed by uncut or classic R-Y reconstruction. Body mass index and blood test were used to evaluate the nutritional status. QOL was evaluated using European Organization for Research and Treatment of Cancer QOL Questionnaire (STO22) and laboratory examinations at postoperative month (POM) 3, 6, 9, and 12. Computed tomography scanning was used to evaluate the skeletal muscle index (SMI) at POM 6 and 12. Endoscopy was performed at POM 12. RESULTS: Operation time, blood loss, time to recovery, complication morbidities, and overall survival were similar between the two groups. Compared with the classic R-Y group, the uncut R-Y group displayed a significantly decreased QOL at POM 9, possibly due to loop recanalization, determined to be occupied 34.2% of the uncut R-Y group. Post-exclusion of recanalization, the QOL was still higher in the classic R-Y group than in the uncut R-Y group, despite their hemoglobin and total protein levels being better than those in the classic R-Y group. Preoperative pre-albumin level and impaired fasting glycemia significantly correlated with the postoperative recanalization. CONCLUSION: We found no significant benefit of uncut over classic R-Y reconstruction which challenges the superiority of the uncut R-Y reconstruction. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02644148.

Optimal postoperative surveillance strategies for cancer survivors with gastric neuroendocrine carcinoma based on individual risk: a multicenter real-world cohort study.

BACKGROUND: The best follow-up strategy for cancer survivors after treatment should balance the effectiveness and cost of disease detection while detecting recurrence as early as possible. Due to the low incidence of gastric neuroendocrine carcinoma and mixed adenoneuroendocrine carcinoma [G-(MA)NEC], high-level evidence-based follow-up strategies is limited. Currently, there is a lack of consensus among clinical practice guidelines regarding the appropriate follow-up strategies for patients with resectable G-(MA)NEC. MATERIALS AND METHODS: The study included patients diagnosed with G-(MA)NEC from 21 centers in China. The random forest survival model simulated the monthly probability of recurrence to establish an optimal surveillance schedule maximizing the power of detecting recurrence at each follow-up. The power and cost-effectiveness were compared with the National Comprehensive Cancer Network, European Neuroendocrine Tumor Society, and European Society for Medical Oncology Guidelines. RESULTS: A total of 801 patients with G-(MA)NEC were included. The patients were stratified into four distinct risk groups utilizing the modified TNM staging system. The study cohort comprised 106 (13.2%), 120 (15.0%), 379 (47.3%), and 196 cases (24.5%) for modified groups IIA, IIB, IIIA, and IIIB, respectively. Based on the monthly probability of disease recurrence, the authors established four distinct follow-up strategies for each risk group. The total number of follow-ups 5 years after surgery in the four groups was 12, 12, 13, and 13 times, respectively. The risk-based follow-up strategies demonstrated improved detection efficiency compared to existing clinical guidelines. Further Markov decision-analytic models verified that the risk-based follow-up strategies were better and more cost-effective than the control strategy recommended by the guidelines. CONCLUSIONS: This study developed four different monitoring strategies based on individualized risks for patients with G-(MA)NEC, which may improve the detection power at each visit and were more economical, effective. Even though our results are limited by the biases related to the retrospective study design, we believe that, in the absence of a randomized clinical trial, our findings should be considered when recommending follow-up strategies for G-(MA)NEC.

Prognostic value of preoperative immune-nutritional scoring systems in remnant gastric cancer patients undergoing surgery.

BACKGROUND: Remnant gastric cancer (GC) is defined as GC that occurs five years or more after gastrectomy. Systematically evaluating the preoperative immune and nutritional status of patients and analyzing its prognostic impact on postoperative remnant gastric cancer (RGC) patients are crucial. A simple scoring system that combines multiple immune or nutritional indicators to identify nutritional or immune status before surgery is necessary. AIM: To evaluate the value of preoperative immune-nutritional scoring systems in predicting the prognosis of patients with RGC. METHODS: The clinical data of 54 patients with RGC were collected and analyzed retrospectively. Prognostic nutritional index (PNI), controlled nutritional status (CONUT), and Naples prognostic score (NPS) were calculated by preoperative blood indicators, including absolute lymphocyte count, lymphocyte to monocyte ratio, neutrophil to lymphocyte ratio, serum albumin, and serum total cholesterol. Patients with RGC were divided into groups according to the immune-nutritional risk. The relationship between the three preoperative immune-nutritional scores and clinical characteristics was analyzed. Cox regression and Kaplan-Meier analysis was performed to analyze the difference in overall survival (OS) rate between various immune-nutritional score groups. RESULTS: The median age of this cohort was 70.5 years (ranging from 39 to 87 years). No significant correlation was found between most pathological features and immune-nutritional status (P > 0.05). Patients with a PNI score < 45, CONUT score or NPS score ≥ 3 were considered to be at high immune-nutritional risk. The areas under the receiver operating characteristic curves of PNI, CONUT, and NPS systems for predicting postoperative survival were 0.611 [95% confidence interval (CI): 0.460-0.763; P = 0.161], 0.635 (95%CI: 0.485-0.784; P = 0.090), and 0.707 (95%CI: 0.566-0.848; P = 0.009), respectively. Cox regression analysis showed that the three immune-nutritional scoring systems were significantly correlated with OS (PNI: P = 0.002; CONUT: P = 0.039; NPS: P < 0.001). Survival analysis revealed a significant difference in OS between different immune-nutritional groups (PNI: 75 mo vs 42 mo, P = 0.001; CONUT: 69 mo vs 48 mo, P = 0.033; NPS: 77 mo vs 40 mo, P < 0.001). CONCLUSION: These preoperative immune-nutritional scores are reliable multidimensional prognostic scoring systems for predicting the prognosis of patients with RGC, in which the NPS system has relatively effective predictive performance.

Proximal gastrectomy may be a reasonable choice for patients with selected proximal advanced gastric cancer: A propensity score-matched analysis.

BACKGROUND: Whether proximal gastrectomy (PG) can be applied to patients with proximal advanced gastric cancer (AGC) remains controversial. We aimed to explore the oncological safety of PG for proximal AGC in this study. METHODS: 452 patients undergoing surgery for proximal AGC in the Affiliated Cancer Hospital of Nanjing Medical University were enrolled in this study. 329 patients with AGC were finally analyzed, of which 254 patients underwent total gastrectomy (TG) and 75 patients underwent PG. We used propensity score-matched (PSM) analysis to reduce biases. RESULTS: After PSM, 67 patients with proximal AGC were included in the PG group and TG group, respectively. The estimated 5-year OS rates for TG and PG group after PSM were 64.3% and 74.9%, respectively, and no significant difference in OS existed between the two groups (p = 0.275). Multivariate analysis showed that PG was not an independent prognostic factor. Incidence of metastasis in No.5 or 6 lymph node (LN) station was significantly higher in the patients with pathological T4 and Borrmann III tumors (9.9% and 10.6%) than those with pathological T2/3 and Borrmann I/II tumors (2.2% and 3.3%). No metastasis was observed in No.5 or 6 LN station in patients with pathological T2/3 tumors or Borrmann I/II tumors when tumor size was ≤4 cm. CONCLUSIONS: PG is a reasonable choice for patients with selected proximal AGC, especially for those with tumors of size ≤4 cm, Borrmann type I/II, and pathological T2/3. Future prospective randomized trials should be conducted first in patients with these specific proximal tumors.

Reappraise role of No. 10 lymphadenectomy for proximal gastric cancer in the era of minimal invasive surgery during total gastrectomy: a pooled analysis of 4 prospective trial.

BACKGROUND: For patients with locally advanced proximal gastric cancer (LAPGC), the individualized selection of patients with highly suspected splenic hilar (No. 10) lymph node (LN) metastasis to undergo splenic hilar lymphadenectomy, is a clinical dilemma. This study aimed to re-evaluate the feasibility and safety of laparoscopic spleen-preserving splenic hilar lymphadenectomy (LSPSHL) and to identify the population who would benefit from it. METHODS: A total of 1068 patients (D2 group = 409; D2 + No. 10 group = 659) who underwent laparoscopic total gastrectomy from four prospective trials between January 2015 and July 2019 were analyzed. RESULTS: No significant difference in the incidence (16.9% vs. 16.4%; P = 0.837) of postoperative complications were found between the two groups. The metastasis rate of No. 10 LN among patients in the D2 + No. 10 group was 10.3% (68/659). Based on the decision tree, patients with LAPGC with tumor invading the greater curvature (Gre), patients with non-Gre-invading LAPGC with a tumor size > 5 cm and clinical positive locoregional LNs were defined as the high-priority No. 10 dissection group. The metastasis rate of No. 10 LNs in the high-priority group was 19.4% (41/211). In high-priority group, the 3-year overall survival of the D2 + No. 10 group was better than that of the D2 group (74.4% vs. 42.1%; P = 0.005), and the therapeutic index of No. 10 was higher than the indices of most suprapancreatic stations. CONCLUSIONS: LSPSHL for LAPGC is safe and feasible when performed by experienced surgeons. LSPSHL could be recommended for the high-priority group patients even without invasion of the Gre.

Safety and feasibility of laparoscopic spleen-preserving No. 10 lymph node dissection for locally advanced upper third gastric cancer: a prospective, multicenter clinical trial.

BACKGROUND: Previous retrospective studies have shown that laparoscopic spleen-preserving D2 total gastrectomy (LSTG) for advanced upper third gastric cancer (AUTGC) is safe. However, all previous studies were underpowered. We therefore conducted a prospective, multicenter study to evaluate the technical safety and feasibility of LSTG for patients with AUTGC. METHODS: Patients diagnosed with AUTGC (cT2-4a, N-/+, M0) underwent LSTG at 19 institutions between September 2016 and October 2017 were included. The number of No. 10 lymph node (LN) dissections, metastasis rates, intraoperative and postoperative complications were investigated. RESULTS: A total of 251 patients were enrolled in the study, and 242 patients were eligible for the per protocol analysis. The average numbers of No. 10 LN dissections and metastases were 2.4 and 0.1, respectively. Eighteen patients (7.4%) had No. 10 LN metastases, and among patients with advanced gastric cancer, the rate of No. 10 LN metastasis was 8.1% (18/223). pN3 status was an independent risk factor for No. 10 LN metastasis. Intraoperative complications occurred in 7 patients, but no patients required conversion to open surgery or splenectomy. The overall postoperative complication rate was 13.6% (33/242). The major complication and mortality rates were 3.3% (8/242) and 0.4% (1/242), respectively. The number of retrieved No. 10 LNs, No. 10 LN metastasis and TNM stage had no significant influence on postoperative complication rates. CONCLUSION: LSTG for AUTGC was safe and effective when performed by very experienced surgeons, this technique could be used in patients who needed splenic hilar lymph node dissection.

Uncut Roux-en-Y Reconstruction in a Laparoscopic Distal Gastrectomy: A Single-Center Study of 228 Consecutive Cases and Short-Term Outcomes.

Aims. We have established a procedure for uncut Roux-en-Y gastrojejunostomy after laparoscopic distal gastrectomy. This study aimed to evaluate the safety and technical feasibility of the procedure for patients with distal gastric cancer according to the short-term outcomes. Methods. Two hundred and twenty-eight consecutive patients who underwent a laparoscopic distal gastrectomy with uncut Roux-en-Y gastrojejunostomy from September 2014 to August 2018 were reviewed retrospectively. All the laparoscopic operations were performed successfully without conversion to open surgery. Results. The mean operative duration was 178.28 ± 32.82 minutes, the mean anastomotic process duration was 28.22 ± 7.50 minutes, the average blood loss was 48.97 ± 29.16 mL, and the overall number of lymph nodes harvested was 37.16 ± 11.47. The mean time of out-of-bed ambulation, anal exsufflation, liquid-diet intake, and duration of hospital stay were 41.99 ± 18.37 hours, 69.57 ± 23.17 hours, 5.06 ± 1.09 days, and 8.77 ± 2.42 days, respectively. Fifteen patients suffered postoperative complications, and the overall incidence rate was 6.58% (15/228). Seventeen patients experienced afferent recanalization, the mean time of which was 11 months after the operation. Conclusion. The laparoscopic uncut Roux-en-Y reconstruction is safe and technically feasible, and it has inspiring short-term outcomes for patients undergoing distal gastrectomy.

Comparison of treatment outcomes between laparoscopic and endoscopic surgeries for relatively small gastric gastrointestinal stromal tumors.

BACKGROUND: Complete surgical resection remains the predominant treatment modality for primary gastrointestinal stromal tumors (GISTs). No therapeutic consensus exists for 2-5 cm gastric GISTs. We compared the efficacy, safety, and prognosis of laparoscopic and endoscopic surgeries in the treatment of relatively small (2-5 cm) intraluminal gastric GISTs. METHODS: We collected 101 patients with relatively small intraluminal gastric GISTs who had integrated clinicopathological data and underwent laparoscopic or endoscopic resection (laparoscopic group n = 66; endoscopic group n = 35). Clinicopathological characteristics, perioperative data, and long-term oncological outcomes were retrospectively analyzed. Comparative analysis of clinicopathological data in the two groups was performed by using a chi-square test, Fisher's exact test, and Student's t-test. Recurrence-free survival (RFS) was analyzed by the log-rank test. RESULTS: All clinicopathological characteristics had no significant difference between the two groups. Patients in the endoscopic group had shorter operation time (P < 0.001), postoperative hospital stay (P < 0.001), time to a liquid diet (P < 0.01), and time to a semi-liquid diet (P < 0.01), and lower hospital charges (P < 0.001), compared to those in the laparoscopic group. Four patients (6.1%) in the laparoscopic group and one patient (2.9%) in the endoscopic group had perioperative complications, but with no significant difference. Recurrence occurred in 6 patients (9.1%) and 2 patients (5.7%) in the laparoscopic and endoscopic groups, respectively. There was no significant difference in RFS between the two groups. CONCLUSION: Endoscopic resection is a feasible and safe treatment modality for patients with relatively small (2-5 cm) intraluminal gastric GISTs. Due to faster recovery and lower cost, endoscopic resection is more suitable for elderly and weak patients, or patients with a poor financial situation.

miR-324-3p promotes gastric cancer development by activating Smad4-mediated Wnt/beta-catenin signaling pathway.

BACKGROUND: Emerging evidence suggested that miRNAs can function as oncogenes or tumor suppressors by regulating downstream target genes. miR-324-3p has been reported to function in several carcinomas, but its role in gastric cancer (GC) is still unknown. This study aims to explore the effects of miR-324-3p on the development of GC. METHODS: Expression of miR-324-3p was examined in GC cells and tissues by qRT-PCR. Effects of miR-324-3p on GC cells were evaluated by cell vitality assay, colony formation assay, cell migration assay, and flow cytometric assay. The dual luciferase assay was used to verify whether miR-324-3p could interact with the potential target genes. Western blot was used to assess the expression level of Smad4 and beta-catenin. Intracellular ATP level was also examined. The tumor xenografts were established using nude mice. A gastric organoid model was made from fresh stomach tissue. RESULTS: miR-324-3p was expressed at higher levels in the tumor tissues compared with adjacent normal tissues. Overexpression of miR-324-3p promoted cell growth, migration, and decreased apoptosis. miR-324-3p repressed the expression of Smad4, and loss of Smad4 activated the Wnt/beta-catenin signaling pathway. Overexpression of Smad4 rescued the effects of miR-324-3p on GC cells. The intracellular ATP level was upregulated with overexpression of miR-324-3p. miR-324-3p facilitated tumor cell colonization and growth in vivo and contributed to the growth of gastric organoids. CONCLUSIONS: The results suggested that miR-324-3p promoted GC through activating the Smad4-mediated Wnt/beta-catenin signaling pathway. The miR-324-3p/Smad4/Wnt signaling axis may be a potential therapeutic target to prevent GC progression.

NIDO, AMOP and vWD domains of MUC4 play synergic role in MUC4 mediated signaling.

MUC4 mucin is well known as an important potential target to overcome pancreatic cancer. Three unique domains (NIDO, AMOP, and vWD) with unclear roles only present in MUC4 but are not found in other membrane-bound mucins. Our previous studies first reported that its splice variant, MUC4/Y can be a model of MUC4 (MUC4 gene fragment is more than 30KB, too huge to clone and eukaryotic express) in pancreatic cancer. More importantly, based on MUC4/Y with the appropriate length of gene sequence, it is easy to construct the unique domain-lacking models of MUC4/Y (MUC4) for research. The present study focuses on investigation of the respective role of the unique NIDO, AMOP, and vWD domain or their synergistic effect on MUC4(MUC4/Y)-mediated functions and mechanisms by series of in vitro assays, sequence-based transcriptome analysis, validation of qRT-PCR & Western blot, and systematic comparative analysis. Our results demonstrate: 1) NIDO, AMOP, and vWD domain or their synergy play significant roles on MUC4/Y-mediated malignant function of pancreatic cancer, downstream of molecule mechanisms, particularly MUC4/Y-triggered malignancy-related positive feedback loops, respectively. 2) The synergistic roles of three unique domains on MUC4/Y-mediated functions and mechanisms are more prominent than the respective domain because the synergy of three domain plays the more remarkable effects on MUC4/Y-mediated signaling hub. Thus, to improve reversed effects of domain-lacking and break the synergism of domains will contribute to block MUC4/Y(MUC4) triggering various oncogenic signaling pathways.

Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer.

Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells.

The transcriptional regulator Yin Yang-1 (YY1) is a tumor suppressor known to be overexpressed in pancreatic cancer. We found that overexpression of YY1 promoted apoptosis and increased the expression and mitochondrial localization of the pro-apoptotic Bax protein in pancreatic cancer cell lines. Luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays revealed binding of YY1 to the BAX promoter. Moreover, YY1 promoted pancreatic cancer cell apoptosis through Bax transcriptional activation and subsequent translocation of Bax to the mitochondrial membrane, leading to cytochrome c release, and caspase activation.YY1 and BAX are co-expressed in pancreatic cancer tissues and higher BAX expression predicts better outcomes for patients. The ability of YY1 to promote apoptosis in pancreatic cancer cells suggests it may represent a valuable diagnostic and therapeutic target.

PP2A inhibitors arrest G2/M transition through JNK/Sp1- dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21.

Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.

Specific-detection of clinical samples, systematic functional investigations, and transcriptome analysis reveals that splice variant MUC4/Y contributes to the malignant progression of pancreatic cancer by triggering malignancy-related positive feedback loops signaling.

BACKGROUND: MUC4 plays important roles in the malignant progression of human pancreatic cancer. But the huge length of MUC4 gene fragment restricts its functional and mechanism research. As one of its splice variants, MUC4/Y with coding sequence is most similar to that of the full-length MUC4 (FL-MUC4), together with alternative splicing of the MUC4 transcript has been observed in pancreatic carcinomas but not in normal pancreas. So we speculated that MUC4/Y might be involved in malignant progression similarly to FL-MUC4, and as a research model of MUC4 in pancreatic cancer. The conjecture was confirmed in the present study. METHODS: MUC4/Y expression was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific probe in the clinic samples. The effects of MUC4/Y were observed by serial in vitro and in vivo experiments based on stable over-expressed cell model. The underlying mechanisms were investigated by sequence-based transcriptome analysis and verified by qRT-PCR, Western blot and enzyme-linked immunosorbent assays. RESULTS: The detection of clinical samples indicates that MUC4/Y is significantly positive-correlated with tumor invasion and distant metastases. Based on stable forced-expressed pancreatic cancer PANC-1 cell model, functional studies show that MUC4/Y enhances malignant activity in vitro and in vivo, including proliferation under low-nutritional-pressure, resistance to apoptosis, motility, invasiveness, angiogenesis, and distant metastasis. Mechanism studies indicate the novel finding that MUC4/Y triggers malignancy-related positive feedback loops for concomitantly up-regulating the expression of survival factors to resist adverse microenvironment and increasing the expression of an array of cytokines and adhesion molecules to affect the tumor milieu. CONCLUSIONS: In light of the enormity of the potential regulatory circuitry in cancer afforded by MUC4 and/or MUC4/Y, repressing MUC4 transcription, inhibiting post-transcriptional regulation, including alternative splicing, or blocking various pathways simultaneously may be helpful for controlling malignant progression. MUC4/Y- expression model is proven to a valuable tool for the further dissection of MUC4-mediated functions and mechanisms.

Elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates natural killer cell dysfunction.

BACKGROUND: Natural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors. METHODS: NK cells isolated from fresh healthy peripheral blood were co-cultured with normal human pancreatic ductal cells hTERT-HPNE and human pancreatic cancer cell lines SW1990 and BxPc-3 in vitro. Then NK cell function was determined by Flow cytometric analysis of surface receptors and cytotoxic granules in NK cells, NK cell apoptosis and cytotoxicity, and Enzyme-linked immunosorbent assay of cytokines. Expression level of MMP-9, IDO and COX-2 in hTERT-HPNE and SW1990 cells were detected by quantitative RT-PCR. Statistical differences between data groups were determined by independent t-tests using SPSS 19.0 software. RESULTS: Our results showed that NK cell function was significantly downregulated following exposure to pancreatic cancer cells compared to normal pancreatic cells, as demonstrated by lower expressions of activating surface receptors (NKG2D, DNAM-1, NKp30 and NKp46) and cytotoxic granules (Perforin and Granzyme B); decreased secretion of cytokines (TNF-α and IFN-γ); and reduced cytotoxicity against myelogenous leukemia K562 cells. Further investigations revealed that MMP-9 and IDO may be implicated in SW1990 cell-induced NK cell dysfunction by facilitating tumor immune evasion. Blockade by TIMP-1 and/or 1-MT could partially restore NK function. CONCLUSIONS: Taken together, elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates NK cell dysfunction. Our findings could contribute to the development of NK cell-based immunotherapy in patients with pancreatic cancer.

Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism.

BACKGROUND: Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified. METHODS: In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software. RESULTS: We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism. CONCLUSIONS: The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.

PP2A inhibitors suppress migration and growth of PANC-1 pancreatic cancer cells through inhibition on the Wnt/ß-catenin pathway by phosphorylation and degradation of ß-catenin.

Cantharidin is an active constituent of mylabris, a traditional Chinese medicine, and presents strong anticancer activity in various cell lines. Cantharidin is a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A). Our previous studies revealed the prospect of application of cantharidin, as well as other PP2A inhibitors, in the treatment of pancreatic cancer. However, the mechanisms involved in the anticancer effect of PP2A inhibitors have not been fully explored. The Wnt/ß­catenin pathway is involved in cell migration and proliferation and participates in the progression of pancreatic cancer. If ß­catenin is phosphorylated and degraded, the Wnt/ß­catenin pathway is blocked. PP2A dephosphorylates ß­catenin and keeps the Wnt/ß­catenin pathway active. In the present study, we found that PP2A inhibitor treatment induced phosphorylation and degradation of ß­catenin. The suppression on the migration and growth of PANC­1 pancreatic cancer cells could be attenuated by pretreatment with FH535, a ß­catenin pathway inhibitor. Microarray showed that PP2A inhibitor treatment induced expression changes in 13 of 138 genes downstream of the ß­catenin pathway. Real­time PCR further confirmed that FH535 attenuated the expression changes induced by PP2A inhibitors in 6 of these 13 candidate genes. These 6 genes, VEGFB, Dkk3, KRT8, NRP1, Cacnalg and WISP2, have been confirmed to participate in the migration and/or growth regulation in previous studies. Thus, the phosphorylation- and degradation-mediated suppression on ß­catenin participates in the cytotoxicity of PP2A inhibitors. Our findings may provide insight into the treatment of pancreatic cancer using a targeting PP2A strategy.

Pancreatic cancer counterattack: MUC4 mediates Fas-independent apoptosis of antigen-specific cytotoxic T lymphocyte.

Tumor-associated MUC4 mucin has considerable potential as an immunotherapy target for pancreatic cancer. In previous studies, we developed dendritic cell (DC) vaccines which elicited MUC4 antigen-specific cytotoxic T lymphocyte (MS-CTL) response against tumor cells in vitro. Due to the observation that MS-CTL apoptotic rate increased significantly when co-cultured with MUC4+ tumor cells compared with T2 cells, we investigated whether high expression levels of MUC4 in pancreatic cancer cells would have an effect on the significant increase of apoptosis rate of MS-CTLs. First, the adverse influence of regulatory T cells (Tregs) was eliminated by CD8+ T lymphocyte sorting before the induction of MS-CTLs. Then, we constructed clonal MUC4-knockdown HPAC pancreatic cancer sublines with different MUC4 expression for co-incubation system. By utilizing appropriate control to rule out the possible apoptosis-induced pathway of intrinsic activated cell-autonomous death (ACAD) and analogous antigen-dependent apoptosis of CTL (ADAC) in our study system, further analysis of the effect of MUC4 membrane-expression, supernatants and blockade of CTL surface Fas receptor on MS-CTL apoptosis was carried out. The results demonstrated that the level of MUC4 membrane expression strongly positively correlated with MS-CTL apoptosis and the influence of supernatants and Fas-blockade did not significantly correlate with MS-CTL apoptosis. This evidence suggested that there may be a novel counterattack pathway of pancreatic cancer cells, which is a MUC4-mediated, cell contact-dependent and Fas-independent process, to induce CTL apoptosis. Therefore, further exploration and understanding of the potential counterattack mechanisms is beneficial to enhance the efficacy of MUC4 specific tumor vaccines.

Expression of granulocyte colony-stimulating factor receptor in rectal cancer.

AIM: To evaluate whether granulocyte colony-stimulating factor receptor (G-CSFR) expression before preoperative irradiation can predict the radiosensitivity of rectal cancer. METHODS: The expression of G-CSFR was examined, using immunohistochemistry, in biopsy specimens from 126 patients with locally advanced rectal adenocarcinoma before preoperative irradiation. Radiosensitivity was then evaluated according to the Rectal Cancer Regression Grading. Endoscopic inspection was used to detect the tumor area in each patient. General patient information, such as age, gender, lymph node status, tumor size and degree of differentiation was recorded. A statistical analysis was then performed to evaluate the correlation between clinical or pathological parameters and G-CSFR expression in tumors. RESULTS: According to endoscopic inspection, the tumor area ranged from 4 to 48 cm² (median, 15 cm²). Positive G-CSFR immunoreactions (G-CSFR⁺) were observed in 85 specimens, and negative (G-CSFR⁻) in 41. No significant differences were found in age, gender, tumor invasion, lymph node status and tumor size between G-CSFR⁺ and G-CSFR⁻ patients. G-CSFR expression was positively correlated with poor radiotherapy response (58.8% vs 75.6%, P = 0.014, r = 0.219). The proportion of well-differentiated tumors in G-CSFR⁺ and G-CSFR⁻ patients was 24.7% and 36.6%, respectively. Sphincter preservation was observed in 57.6% of G-CSFR⁺ patients and 78.5% of G-CSFR⁻ patients. Significant correlations were found between G-CSFR expression and tumor differentiation (24.7% vs 36.6%, P = 0.019, r = 0.210), as well as sphincter preservation (57.6% vs 78.5%, P = 0.044, r = 0.180). CONCLUSION: The expression of G-CSFR before preoperative irradiation may predict the radiosensitivity of rectal cancer.

Comprehensive analysis of the percentage of surface receptors and cytotoxic granules positive natural killer cells in patients with pancreatic cancer, gastric cancer, and colorectal cancer.

BACKGROUND: Digestive malignancies, especially pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer (CRC), still occur at persistently high rates, and disease progression in these cancers has been associated with tumor immunosurveillance escape. Natural killer (NK) cell dysfunction may be responsible for this phenomenon, however, the exact relationship between tumor immunosurveillance escape in digestive malignancies and NK cell dysfunction remains unclear. METHODS: Percentage of the surface receptors NKG2A, KIR3DL1, NKG2D, NKp30, NKp44, NKp46, and DNAM-1, as well as the cytotoxic granules perforin and granzyme B positive NK cells were determined in patients with pancreatic cancer (n=31), gastric cancer (n=31), and CRC (n=32) prior to surgery and healthy controls (n=31) by multicolor flow cytometry. Independent t-tests or Mann-Whitney U-tests were used to compare the differences between the patient and healthy control groups, as well as the differences between patients with different pathologic features of cancer. RESULTS: Percentage of NKG2D, NKp30, NKp46, and perforin positive NK cells was significantly down-regulated in patients with PC compared to healthy controls, as well as GC and CRC; reduced levels of these molecules was associated with indicators of disease progression in each malignancy (such as histological grade, depth of invasion, lymph node metastasis). On the contrary, percentage of KIR3DL1 positive NK cells was significantly increased in patients with PC, as well as GC and CRC, but was not associated with any indicators of disease progression. CONCLUSIONS: Altered percentage of surface receptors and cytotoxic granules positive NK cells may play a vital role in tumor immunosurveillance escape by inducing NK cell dysfunction in patients with PC, GC, and CRC.