Statins exert anti-growth effects by suppressing YAP/TAZ expressions via JNK signal activation and eliminate the immune suppression by downregulating PD-L1 expression in pancreatic cancer.

Statins are cholesterol-lowering agents that act as inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzymeA (HMG CoA) reductase. Recently, statins have received a lot of attention, especially regarding how statins act on the immune system. Here, the clinical impact of statin intake was examined in patients with resected pancreatic cancer, and the underlying mechanisms were investigated in vitro and in vivo. We found that statin intake was associated with favorable prognostic outcomes in patients with resectable pancreatic cancer. Statins, especially lipophilic statins, exert anti-proliferative effects on pancreatic cancer cells in vitro (simvastatin > fluvastatin > atorvastatin > rosuvastatin > pravastatin). Simvastatin had an anti-proliferative effect on pancreatic cancer cells with decreased the yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression by activating the JNK pathway, and simvastatin treatment with oxaliplatin revealed additive anti-growth effects. Furthermore, lipophilic and hydrophilic statins suppressed programmed cell death ligand 1 (PD-L1) expression by downregulating TAZ. Simvastatin treatment with an anti-PD-1 drug (BP0273) provided immediate anti-growth effects compared to controls, such as anti-PD-1 only and simvastatin only, and suppressed progressive disease during the early period of anti-PD-1 treatment in vivo. In conclusion, Statins display two distinct anti-cancer effects (direct anti-growth effect and elimination of immune suppression by downregulating PD-L1 expression) by targeting YAP/TAZ expression.

Hyperglycaemia induces metabolic reprogramming into a glycolytic phenotype and promotes epithelial-mesenchymal transitions via YAP/TAZ-Hedgehog signalling axis in pancreatic cancer.

BACKGROUND: Hyperglycaemia is a well-known initial symptom in patients with pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming in cancer, described as the Warburg effect, can induce epithelial-mesenchymal transition (EMT). METHODS: The biological impact of hyperglycaemia on malignant behaviour in PDAC was examined by in vitro and in vivo experiments. RESULTS: Hyperglycaemia promoted EMT by inducing metabolic reprogramming into a glycolytic phenotype via yes-associated protein (YAP)/PDZ-binding motif (TAZ) overexpression, accompanied by GLUT1 overexpression and enhanced phosphorylation Akt in PDAC. In addition, hyperglycaemia enhanced chemoresistance by upregulating ABCB1 expression and triggered PDAC switch into pure basal-like subtype with activated Hedgehog pathway (GLI1 high, GATA6 low expression) through YAP/TAZ overexpression. PDAC is characterised by abundant stroma that harbours tumour-promoting properties and chemoresistance. Hyperglycaemia promotes the production of collagen fibre-related proteins (fibronectin, fibroblast activation protein, COL1A1 and COL11A1) by stimulating YAP/TAZ expression in cancer-associated fibroblasts (CAFs). Knockdown of YAP and/or TAZ or treatment with YAP/TAZ inhibitor (K975) abolished EMT, chemoresistance and a favourable tumour microenvironment even under hyperglycemic conditions in vitro and in vivo. CONCLUSION: Hyperglycaemia induces metabolic reprogramming into glycolytic phenotype and promotes EMT via YAP/TAZ-Hedgehog signalling axis, and YAP/TAZ could be a novel therapeutic target in PDAC.

Thrombospondin-1 overexpression stimulates loss of Smad4 and accelerates malignant behavior via TGF-ß signal activation in pancreatic ductal adenocarcinoma.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma and cancer-associated fibroblasts (CAFs) provide a favorable tumor microenvironment. Smad4 is known as tumor suppressor in several types of cancers including PDAC, and loss of Smad4 triggers accelerated cell invasiveness and metastatic potential. The thrombospondin-1 (TSP-1) can act as a major activator of latent transforming growth factor-ß (TGF-ß) in vivo. However, the roles of TSP-1 and the mediator of Smad4 loss and TGF-ß signal activation during PDAC progression have not yet been addressed. The aim is to elucidate the biological role of TSP-1 in PDAC progression. METHODS AND RESULTS: High substrate stiffness stimulated TSP-1 expression in CAFs, and TSP-1 knockdown inhibited cell proliferation with suppressed profibrogenic and activated stroma-related gene expressions in CAFs. Paracrine TSP-1 treatment for PDAC cells promoted cell proliferation and epithelial mesenchymal transition (EMT) with activated TGF-ß signals such as phosphorylated Akt and Smad2/3 expressions. Surprisingly, knockdown of DPC4 (Smad4 gene) induced TSP-1 overexpression with TGF-ß signal activation in PDAC cells. Interestingly, TSP-1 overexpression also induced downregulation of Smad4 expression and enhanced cell proliferation in vitro and in vivo. Treatment with LSKL peptide, which antagonizes TSP-1-mediated latent TGF-ß activation, attenuated cell proliferation, migration and chemoresistance with enhanced apoptosis in PDAC cells. CONCLUSIONS: TSP-1 derived from CAFs stimulates loss of Smad4 expression in cancer cells and accelerates malignant behavior by TGF-ß signal activation in PDAC. TSP-1 could be a novel therapeutic target, not only for CAFs in stiff stroma, but also for cancer cells in the PDAC microenvironment.

Western-Style Diet, pks Island-Carrying Escherichia coli, and Colorectal Cancer: Analyses From Two Large Prospective Cohort Studies.

BACKGROUND & AIMS: Evidence supports a carcinogenic role of Escherichia coli carrying the pks island that encodes enzymes for colibactin biosynthesis. We hypothesized that the association of the Western-style diet (rich in red and processed meat) with colorectal cancer incidence might be stronger for tumors containing higher amounts of pks+E coli. METHODS: Western diet score was calculated using food frequency questionnaire data obtained every 4 years during follow-up of 134,775 participants in 2 United States-wide prospective cohort studies. Using quantitative polymerase chain reaction, we measured pks+E coli DNA in 1175 tumors among 3200 incident colorectal cancer cases that had occurred during the follow-up. We used the 3200 cases and inverse probability weighting (to adjust for selection bias due to tissue availability), integrated in multivariable-adjusted duplication-method Cox proportional hazards regression analyses. RESULTS: The association of the Western diet score with colorectal cancer incidence was stronger for tumors containing higher levels of pks+E coli (Pheterogeneity = .014). Multivariable-adjusted hazard ratios (with 95% confidence interval) for the highest (vs lowest) tertile of the Western diet score were 3.45 (1.53-7.78) (Ptrend = 0.001) for pks+E coli-high tumors, 1.22 (0.57-2.63) for pks+E coli-low tumors, and 1.10 (0.85-1.42) for pks+E coli-negative tumors. The pks+E coli level was associated with lower disease stage but not with tumor location, microsatellite instability, or BRAF, KRAS, or PIK3CA mutations. CONCLUSIONS: The Western-style diet is associated with a higher incidence of colorectal cancer containing abundant pks+E coli, supporting a potential link between diet, the intestinal microbiota, and colorectal carcinogenesis.

Relationship between Fusobacterium nucleatum and antitumor immunity in colorectal cancer liver metastasis.

Fusobacterium nucleatum has been detected in 8%-13% of human colorectal cancer, and shown to inhibit immune responses against primary colorectal tumors in animal models. Thus, we hypothesized that the presence of F. nucleatum might be associated with reduced T cell density in colorectal cancer liver metastases (CRLM). We quantified F. nucleatum DNA in 181 CRLM specimens using quantitative PCR assay. The densities of CD8+ T cells, CD33+ cells (marker for myeloid-derived suppressor cells [MDSCs]), and CD163+ cells (marker for tumor-associated macrophages [TAMs]) in CRLM tissue were determined by immunohistochemical staining. Fusobacterium nucleatum was detected in eight (4.4%) of 181 CRLM specimens. Compared with F. nucleatum-negative CRLM, F. nucleatum-positive CRLM showed significantly lower density of CD8+ T cells (P = .033) and higher density of MDSCs (P = .001). The association of F. nucleatum with the density of TAMs was not statistically significant (P = .70). The presence of F. nucleatum is associated with a lower density of CD8+ T cells and a higher density of MDSCs in CRLM tissue. Upon validation, our findings could provide insights to develop strategies that involve targeting microbiota and immune cells for the prevention and treatment of CRLM.

High ARHGEF2 (GEF-H1) Expression is Associated with Poor Prognosis Via Cell Cycle Regulation in Patients with Pancreatic Cancer.

BACKGROUND: Pancreatic cancer has an extremely poor prognosis, even after curative resection. Treatment options for pancreatic cancer remain limited, therefore new therapeutic targets are urgently needed. We searched for genes predictive of poor prognosis in pancreatic cancer using a public database and validated the survival impact of the selected gene in a patient cohort. METHODS: We used a public database to search for genes associated with early pancreatic cancer recurrence. As a validation cohort, 201 patients who underwent radical resection in our institution were enrolled. Expression of the target gene was evaluated using immunohistochemistry (IHC). We evaluated growth and invasiveness using small interfering RNAs, then performed pathway analysis using gene set enrichment analysis. RESULTS: We extracted ARHGEF2 from GSE21501 as a gene with a high hazard ratio (HR) for early recurrence within 1 year. The high ARHGEF2 expression group had significantly poorer recurrence-free survival (RFS) and poorer overall survival (OS) than the low ARHGEF2 expression group. Multivariate analysis demonstrated that high ARHGEF2 expression was an independent poor prognostic factor for RFS (HR 1.92) and OS (HR 1.63). In vitro, ARHGEF2 suppression resulted in reduced cell growth and invasiveness. Bioinformatic analysis revealed that ARHGEF2 expression was associated with MYC, G2M, E2F, and CDC25A expression, suggesting that c-Myc and cell cycle genes are associated with high ARHGEF2 expression. IHC revealed a positive correlation between ARHGEF2 and c-Myc expression. CONCLUSIONS: High ARHGEF2 expression is associated with cell cycle progression, and predicts early recurrence and poor survival in patients with pancreatic cancer.

Immunogenic characteristics of microsatellite instability-low esophagogastric junction adenocarcinoma based on clinicopathological, molecular, immunological and survival analyses.

Microsatellite instability (MSI) is categorized by mutation frequency: high MSI (MSI-H), low MSI (MSI-L) and microsatellite stable (MSS). MSI-H tumors have a distinct immunogenic phenotype, with immunotherapies using checkpoint inhibitors already approved for the treatment of MSI-H gastroesophageal adenocarcinoma (GEA); this is not observed for MSI-L or MSS. Here, we tested the hypothesis that MSI-L tumors are also a distinct phenotype and potentially immunogenic. MSI-PCR assays (BAT25, BAT26, BAT40, D2S123, D5S346 and D17S250) were performed on 363 Epstein-Barr virus-negative, surgically resected esophagogastric junction (EGJ) adenocarcinoma samples. Tumors were characterized as MSI-H (≥2 markers), MSI-L (1 marker) or MSS (0 markers). CD8+ cell counts, PD-L1 and HER2 expression levels, TP53 mutations, epigenetic alterations and prognostic significance were also examined. All pathological and molecular experiments were conducted using serial, whole-tumor sections of chemo-naïve surgical specimens. MSI-H and MSI-L were assigned to 28 (7.7%) and 24 (6.6%) cases, respectively. Compared to MSS cases, MSI-L cases had significantly higher intratumoral CD8+ cell infiltration (P = .048) and favorable EGJ cancer-specific survival (multivariate hazard ratio = 0.35, 95% CI, 0.12-0.82; P = .012). MSI-L tumors were also significantly associated with TP53-truncating mutations as compared to MSI-H (P = .009) and MSS (P = .012) cases, and this trend was also observed in GEA data from The Cancer Genome Atlas (TCGA). Indel mutational burden among TCGA MSI-L tumors was significantly higher than that of MSS tumors (P = .016). These results suggest that MSI-L tumors may have a distinct tumor phenotype and be potentially immunogenic in EGJ adenocarcinoma.

Mucosal cancer-associated microbes and anastomotic leakage after resection of colorectal carcinoma.

BACKGROUND: Clinical and experimental evidence suggests that colorectal mucosal microbiota changes during colorectal carcinogenesis and may impair colorectal anastomotic wound healing. Thus, we hypothesized that amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with anastomotic leakage after resection for colorectal carcinoma. METHODS: We analyzed 256 fresh frozen tissues of colorectal cancer from patients who underwent elective colorectal resection and anastomosis. Amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, in colorectal cancer tissues were measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable logistic regression analysis was conducted to assess associations of these microbes with anastomotic leakage, adjusting for patient and tumor characteristics, and surgery-related factors. RESULTS: Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Ptrend = 0.004). The association of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with anastomotic leakage was not statistically significant. CONCLUSIONS: The amount of Bifidobacterium genus in colorectal tissue is associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection.

Indoleamine 2, 3-dioxygenase 1 promoter hypomethylation is associated with poor prognosis in patients with esophageal cancer.

Indoleamine 2, 3-dioxygenase 1 (IDO1) is a primary enzyme that generates immunosuppressive metabolites. It plays a major role in tumor immunology and is a potential immune-based therapeutic target. We have reported that IDO1 protein expression was associated with an unfavorable clinical outcome in esophageal cancer. Recently, it has been reported that IDO1 expression is regulated by methylation of the IDO1 promoter. Thus, the aim of this study was to examine the relationship between IDO1 expression, IDO1 promoter methylation, and clinicopathological features in esophageal cancer. We first confirmed changes in IDO1 expression levels in vitro by treating cells with 5-azacytidine. We then evaluated the relationship between IDO1 expression levels, IDO1 promoter methylation (bisulfite pyrosequencing), and clinicopathological features using 40 frozen samples and 242 formalin-fixed, paraffin-embedded samples resected from esophageal cancer patients. We treated cell lines with 5-azacytidine, and the resulting hypomethylation induced significantly higher IDO1 expression (P < .001). In frozen samples, IDO1 expression levels correlated inversely with IDO1 promoter methylation levels (R = -0.47, P = .0019). Furthermore, patients in the IDO1 promoter hypomethylation group (n = 67) had a poor prognosis compared with those in the IDO1 promoter hypermethylation group (n = 175) (overall survival, P = .011). Our results showed that IDO1 promoter hypomethylation regulated IDO1 expression and was associated with a poor prognosis in esophageal cancer patients.

Glucose transporter 1 regulates the proliferation and cisplatin sensitivity of esophageal cancer.

Glucose transporter 1 (GLUT1) expression is a prognostic marker for esophageal squamous cell carcinoma (ESCC). Recent work on GLUT1 and development of specific inhibitors supports the feasibility of GLUT1 inhibition as a treatment for various cancers. The anti-proliferative effects of GLUT1-specific small interfering RNA (siRNA) and a GLUT1 inhibitor were evaluated in ESCC cell lines. Expression of pro-proliferative and anti-proliferative signaling and effector molecules was examined by western blotting and quantitative RT-PCR. GLUT1 expression in pretreatment clinical biopsy samples was measured by immunohistochemistry and correlated with various clinicopathological parameters and response to chemotherapy. The reduction in standardized uptake value (SUV) of 18 F-fluoro-deoxyglucose was calculated using the formula: ([pretreatment SUVmax  - posttreatment SUVmax ]/pretreatment SUVmax ) × 100. GLUT1-specific siRNA expression in ESCC cells inhibited their proliferation, increased expression of p27kip, and decreased expression of cyclin-dependent kinase 6, pyruvate kinase muscle isozyme M2, lactate dehydrogenase A and phospho-ERK1/2. Suppression of GLUT1 by siRNA increased low-dose cisplatin-induced inhibition of proliferation of TE-11 ESCC cells, which express high GLUT1 levels. Similarly, BAY-876, a GLUT1 inhibitor, enhanced cisplatin-mediated inhibition of ESCC cell proliferation. GLUT1 expression in pretreatment biopsy samples was associated with the response to chemotherapy as well as the pathological tumor stage and histological response grade after esophagectomy. Finally, GLUT1-negative tumors showed a significantly larger reduction in SUVmax (61.2% ± 4.5%) compared with GLUT1-positive tumors (46.2% ± 4.4%). GLUT1 expression may be a surrogate marker of response to chemotherapy, and inhibition of GLUT1 may be a potential novel therapy for ESCC patients.