S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer.

Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFß1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.

Five EMT-related genes signature predicts overall survival and immune environment in microsatellite instability-high gastric cancer.

BACKGROUND: Microsatellite instability-high (MSI-H) subgroup of gastric cancer (GC) is characterized by a high tumor mutational burden, increased lymphocytic infiltration, and enhanced inflammatory cytokines. GC patients with MSI-H status have a good response to immune checkpoint blockade management. However, heterogeneity within the subtype and the underlying mechanisms of shaping tumor microenvironments remain poorly understood. METHODS: RNA expression levels and clinical parameters of GC were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The data were analyzed using single-sample Gene Set Enrichment Analysis (ssGSEA), univariate Cox regression, multivariate Cox regression, and Least Absolute Shrinkage Selection Operator (LASSO) regression. In addition, multiplex immunohistochemistry (mIHC) was used in our clinical cohort for the tumor microenvironment study. RESULTS: By ssGSEA and survival analysis, the EMT signaling pathway was identified as a representative pathway, which can stratify the patients with MSI-H GC with significant survival predictive power. Then, a novel representative EMT-related five-gene signature (namely CALU, PCOLCE2, PLOD2, SGCD, and THBS2) was established from EMT signaling gene set, which sensitivity and specificity were further validated in the independent GEO database (GSE62254) cohort for disease outcome prediction. Based on public single-cell data and in situ immunohistochemistry, we found that most of these five genes were abundantly expressed in cancer-associated fibroblasts. Furthermore, patients with high or low risk divided by this five-gene signature exhibited a strong correlation of the distinct patterns of tumor immune microenvironment. By mIHC staining of sections from 30 patients with MSI-H status, we showed that the patients with better prognoses had the increased infiltration of CD8+ cells in the primary tumoral tissue. CONCLUSION: Our study developed a simple five-gene signature for stratifying MSI-H GC patients with survival predictive power.

CDK8 and CDK19 regulate intestinal differentiation and homeostasis via the chromatin remodeling complex SWI/SNF.

Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. The cyclin dependent kinases CDK8 and CDK19 (Mediator kinases) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinases have been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using genetically defined models and pharmacological inhibitors, we showed that CDK8 and CDK19 function in a redundant manner to regulate intestinal lineage specification in humans and mice. The Mediator kinase module bound and phosphorylated key components of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF) in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator colocalized at distinct lineage-specifying enhancers in a CDK8/19-dependent manner. Thus, these studies reveal a transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodeling complex SWI/SNF and Mediator kinase.

The kinase activity of integrin-linked kinase regulates cellular senescence in gastric cancer.

The activity of integrin-linked kinase (ILK) in cancerous cells is often oncogenic and associated with malignant properties, such as uncontrolled cell cycle progression and evasion from senescence. However, the role of ILK in cellular senescence in gastric cancer (GC) has not been previously examined. We generated single-cell clones of ILK knock-out using CRISPR-Cas9 in human GC lines with mesenchymal or epithelial histology. Cells with no residual ILK expression exhibited strong cellular senescence with diminished clathrin-mediated endocytosis, Surprisingly, ILK loss-induced cellular senescence appeared to be independent of its function in integrin signaling. The low dose of CPD22, a small molecule inhibitor of ILK activity-induced senescence in three GC cell lines with different histologies. Furthermore, senescent cells with ILK depletion transfected with N-terminal truncated ILK mutant remaining catalytic domains displayed the reduction of senescent phenotypes. RNA sequencing and cytokine array results revealed the enrichment of multiple pro-inflammatory signaling pathways in GC lines in the absence of ILK. Our study identified the important role and the potential mechanism of ILK in the cellular senescence of cancerous epithelial cells. The inhibition of ILK activity using small molecule compounds could have a pro-senescent effect as a therapeutic option for GC.

Complete loss of miR-200 family induces EMT associated cellular senescence in gastric cancer.

The EMT (epithelial-to-mesenchymal-transition) subtype of gastric cancer (GC) is associated with poor treatment responses and unfavorable clinical outcomes. Despite the broad physiological roles of the micro-RNA (miR)-200 family, they largely serve to maintain the overall epithelial phenotype. However, during late-stage gastric tumorigenesis, members of the miR-200 family are markedly suppressed, resulting in the transition to the mesenchymal state and the acquisition of invasive properties. As such, the miR-200 family represents a robust molecular marker of EMT, and subsequently, disease severity and prognosis. Most reports have studied the effect of single miR-200 family member knockdown. Here, we employ a multiplex CRISPR/Cas9 system to generate a complete miR-200 family knockout (FKO) to investigate their collective and summative role in regulating key cellular processes during GC pathogenesis. Genetic deletion of all miR-200s in the human GC cell lines induced potent morphological alterations, G1/S cell cycle arrest, increased senescence-associated ß-galactosidase (SA-ß-Gal) activity, and aberrant metabolism, collectively resembling the senescent phenotype. Coupling RNA-seq data with publicly available datasets, we revealed a clear separation of senescent and non-senescent states amongst FKO cells and control cells, respectively. Further analysis identified key senescence-associated secretory phenotype (SASP) components in FKO cells and a positive feedback loop for maintenance of the senescent state controlled by activation of TGF-ß and TNF-α pathways. Finally, we showed that miR-200 FKO associated senescence in cancer epithelial cells significantly recruited stromal cells in the tumor microenvironment. Our work has identified a new role of miR-200 family members which function as an integrated unit serving to link senescence with EMT, two major conserved biological processes.

The EUS molecular evaluation of pancreatic cancer: A prospective multicenter cohort trial.

BACKGROUND AND OBJECTIVES: Patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (A-PDAC) are not candidates for surgical resection and are often offered palliative chemotherapy. The ready availability of a safe and effective tumor sampling technique to provide material for both diagnosis and comprehensive genetic profiling is critical for informing precision medicine in A-PDAC, thus potentially increasing survival. The aim of this study is to examine the feasibility and benefits of routine comprehensive genomic profiling (CGP) of A-PDAC using EUS-FNA material. METHODS: This is a prospective cohort study to test the clinical utility of fresh frozen or archival EUS-FNA samples in providing genetic material for CGP. The results of the CGP will be reviewed at a molecular tumor board. The proportion of participants that have a change in their treatment recommendations based on their individual genomic profiling will be assessed. Correlations between CGP and stage, prognosis, response to treatment and overall survival will also be investigated. This study will open to recruitment in 2020, with a target accrual of 150 A-PDAC patients within 36 months, with a 2-year follow-up. It is expected that the majority of participants will be those who have already consented for their tissue to be biobanked in the Victorian Pancreatic Cancer Biobank at the time of diagnostic EUS-FNA. Patients without archival or biobanked material that is suitable for CGP may be offered a EUS-FNA procedure for the purposes of obtaining fresh frozen material. DISCUSSION: This trial is expected to provide crucial data regarding the feasibility of routine CGP of A-PDAC using EUS-FNA material. It will also provide important information about the impact of this methodology on patients' survival.

Targeted Transcriptome and KRAS Mutation Analysis Improve the Diagnostic Performance of EUS-FNA Biopsies in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis, and current diagnostic tests have suboptimal sensitivity. Incorporating standard cytology with targeted transcriptomic and mutation analysis may improve upon the accuracy of diagnostic biopsies, thus reducing the burden of repeat procedures and delays to treatment initiation. EXPERIMENTAL DESIGN: We reviewed the accuracy of 308 endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) diagnostic PDAC biopsies using a large multicenter clinical and biospecimen database, then performed RNA sequencing on 134 EUS-FNA biopsies spanning all stages of disease. We identified a transcriptomic diagnostic gene signature that was validated using external datasets and 60 further diagnostic EUS-FNAs. KRAS digital droplet PCR (ddPCR) analysis was performed and correlated with signature gene expression. RESULTS: The sensitivity of EUS-FNA cytology in diagnosing solid pancreatic masses in our retrospective cohort of 308 patients was 78.6% (95% confidence interval, 73.2%-83.2%). KRAS mutation analysis and our custom transcriptomic signature significantly improved upon the diagnostic accuracy of standard cytology to 91.3% in external validation sets and 91.6% in our validation cohort (n = 60). Exploratory ddPCR analysis of KRAS-mutant allele fraction (MAF%) correlated closely to signature performance and may represent a novel surrogate marker of tumor cellularity in snap-frozen EUS-FNA biopsies. CONCLUSIONS: Our findings support snap-frozen EUS-FNA biopsies as a feasible tissue source for the integrated genomic and transcriptomic analysis of patients presenting with PDAC from all tumor stages, including cases with nondiagnostic cytology. Our transcriptome-derived genetic signature in combination with tissue KRAS mutation analysis significantly improves upon the diagnostic accuracy of current standard procedures, and has potential clinical utility in improving the speed and accuracy of diagnosis for patients presenting with PDAC.

TLR2 activation promotes tumour growth and associates with patient survival and chemotherapy response in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.