KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

KDM6A Loss Recruits Tumor-Associated Neutrophils and Promotes Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, the impact of KDM6A loss on the PDAC tumor immune microenvironment is not known. This study used a genetically engineered, pancreas-specific Kdm6a knockout (KO) PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased tumor-associated neutrophils and neutrophil extracellular traps (NET) formation, which are known to contribute to PDAC progression. Genome-wide bromouridine sequencing analysis to evaluate nascent RNA synthesis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), was upregulated in KDM6A KO PDAC cells. KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruited neutrophils. Furthermore, in a syngeneic orthotopic mouse model, treatment with a CXCL1 neutralizing antibody blocked the chemotactic and NET-promoting properties of KDM6A-deficient PDAC cells and suppressed tumor growth, confirming CXCL1 as a key mediator of chemotaxis and PDAC growth driven by KDM6A loss. These findings shed light on how KDM6A regulates the tumor immune microenvironment and PDAC progression and suggests that the CXCL1-CXCR2 axis may be a candidate target in PDAC with KDM6A loss. SIGNIFICANCE: KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1-CXCR2 signaling axis in tumors with low KDM6A.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development. METHODS: We performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis. RESULTS: Loss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial-mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata. CONCLUSIONS: Loss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

Neutrophil in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a poor prognosis and low survival rates. PDAC is characterized by a fibroinflammatory tumor microenvironment enriched by abundant fibroblasts and a variety of immune cells, contributing to its aggressiveness. Neutrophils are essential infiltrating immune cells in the PDAC microenvironment. Recent studies have identified several cellular mechanisms by which neutrophils are recruited to tumor lesion and promote tumorigenesis. This review summarizes the current understanding of the interplay between neutrophils, tumor cells, and other components in the PDAC tumor microenvironment. The prognosis and therapeutic implications of neutrophils in PDAC are also discussed.

Extracellular Trap-Mimicking DNA-Histone Mesostructures Synergistically Activate Dendritic Cells.

Extracellular traps (ETs), such as neutrophil extracellular traps, are a physical mesh deployed by immune cells to entrap and constrain pathogens. ETs are immunogenic structures composed of DNA, histones, and an array of variable protein and peptide components. While much attention has been paid to the multifaceted function of these structures, mechanistic studies of ETs remain challenging due to their heterogeneity and complexity. Here, a novel DNA-histone mesostructure (DHM) formed by complexation of DNA and histones into a fibrous mesh is reported. DHMs mirror the DNA-histone structural frame of ETs and offer a facile platform for cell culture studies. It is shown that DHMs are potent activators of dendritic cells and identify both the methylation state of DHMs and physical interaction between dendritic cells and DHMs as key tuning switches for immune stimulation. Overall, the DHM platform provides a new opportunity to study the role of ETs in immune activation and pathophysiology.

p38-mediated phosphorylation at T367 induces EZH2 cytoplasmic localization to promote breast cancer metastasis.

Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. Here we show that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. p38-mediated EZH2 phosphorylation at T367 promotes EZH2 cytoplasmic localization and potentiates EZH2 binding to vinculin and other cytoskeletal regulators of cell migration and invasion. Ectopic expression of a phospho-deficient T367A-EZH2 mutant is sufficient to inhibit EZH2 cytoplasmic expression, disrupt binding to cytoskeletal regulators, and reduce EZH2-mediated adhesion, migration, invasion, and development of spontaneous metastasis. These results point to a PRC2-independent non-canonical mechanism of EZH2 pro-metastatic function.

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels.

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDHhighCD44high) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p<0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo. Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels.

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas.

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal.

Cancer-stromal cell interactions are a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, have limitations in three aspects: 1) limited control of the cell microenvironment; 2) inability to study cell behavior in a single-cell manner; and 3) have difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is for improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells while maintaining nutrient renewal. In this work, two-phase flow was used as a simple isolation method, separating the microenvironment of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect the cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows area reduction of vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between the cell culture layer and the media exchange layer. The selectivity of the semi-permeable membrane results in the retention of the signaling proteins within the chamber while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk between cells in other chambers. Utilizing these innovations, we co-cultured UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to simulate tumor proliferation enhancement in the vascular endothelial niche.

In vitro time-kill studies of antimicrobial agents against blood isolates of imipenem-resistant Acinetobacter baumannii, including colistin- or tigecycline-resistant isolates.

The emergence of colistin or tigecycline resistance as well as imipenem resistance in Acinetobacter baumannii poses a great therapeutic challenge. The bactericidal and synergistic effects of several combinations of antimicrobial agents against imipenem-, colistin- or tigecycline-resistant A. baumannii isolates were investigated by in vitro time-kill experiments. Six imipenem-resistant A. baumannii blood isolates were examined in this study, including colistin- and tigecycline-susceptible, colistin-resistant but tigecycline-susceptible, and colistin-susceptible but tigecycline-resistant isolates. Time-kill studies were performed using five antimicrobial agents singly or in combinations (imipenem plus colistin, imipenem plus ampicillin-sulbactam, colistin plus rifampicin, colistin plus tigecycline, and tigecycline plus rifampicin) at concentrations of 0.5× and 1× their MICs. Only imipenem was consistently effective as a single agent against all six A. baumannii isolates. Although the effectiveness of combinations of 0.5× MIC antimicrobial agents was inconsistent, combination regimens using 1× MIC of the antimicrobial agents displayed excellent bactericidal activities against all six A. baumannii isolates. Among the combinations of 0.5× MIC antimicrobial agents, the combination of colistin and tigecycline showed synergistic or bactericidal effects against four of the isolates. This in vitro time-kill analysis suggests that antimicrobial combinations are effective for killing imipenem-resistant A. baumannii isolates, even if they are simultaneously resistant to either colistin or tigecycline. However, the finding that the combinations of 0.5× MIC antimicrobial agents were effective on only some isolates may warrant further investigation of the doses of combination agents needed to kill resistant A. baumannii.

Establishment and characterization of 13 human colorectal carcinoma cell lines: mutations of genes and expressions of drug-sensitivity genes and cancer stem cell markers.

Thirteen human colorectal cancer (CRC) cell lines were established from 10 primary tumors and 3 metastatic tumors obtained from 13 Korean patients. Characteristics of the cell lines including morphology in vivo and in vitro; mutations of the K-ras, p53, APC and MMR genes and microsatellite instability (MSI) status in vitro were determined. Expression of drug-sensitivity genes including MDR1, MXR, MRP1 and COX2 was also analyzed. The cell lines were unique as judged by DNA fingerprinting using 16 short tandem repeats. Eleven of the cell lines grew as adherent populations and the remaining two as floating aggregates. None of the cell lines were contaminated with Mycoplasma or bacteria. All cell lines showed high viability with relatively long doubling times. Six cell lines contained mutations at K-ras. Seven cell lines displayed p53 gene missense, nonsense and frameshift mutations. MSI was found in three cell lines and two cell lines with an MSI-high phenotype-possessed hMLH1 mutations. Nine cell lines had an APC mutation. MRP1 was highly expressed in all cell lines, and high expression of MDR1, MXR and COX2 evident in eight, six and six cell lines, respectively. Embryonal stem cell markers (MELK, SOX4 and OCT4) were expressed in most of cell lines. The cancer stem cell biomarkers CD133, CD44 and Lgr5 were expressed in 12, 13 and 13 cell lines, respectively. The presently well-characterized CRC cell lines should be useful in investigations of the biological characteristics of CRC, particularly for investigations related to gene alterations associated with CRC and biology of cancer stem cells.

Effects of pneumoperitoneum and position changes on blood pressure variability and heart rate variability during laparoscopy-assisted vaginal hysterectomy.

BACKGROUND: This study was designed to assess the effects of pneumoperitoneum and positional changes on the autonomic nervous system (ANS) in laparoscopy-assisted vaginal hysterectomy (LAVH) patients. METHODS: Systolic blood pressures and R-R interval were recorded for 5 minutes in 22 patients, and then power spectral analyses were conducted to evaluate the ANS. The following variables were measured at various positions: preinduction (BASE), prepneumoperitoneum (PREPP), pneumoperitoneum at head-down (PP), normoperitoneum at supine (POSTPP). RESULTS: High frequency of heart rate variability (HRVHF), Low frequency of heart rate variability (HRVLF), Low frequency of blood pressure variability (BPVLF), LF/HF ratios of HRV (LFHFr) were significantly lower than that of BASE at PREPP. HRVHF, HRVLF, BPVLF were significantly lower than that of BASE at PP. At PP, normalized HF of HRV (nuHF) is significantly lower than that of BASE and normalized LF of HRV (nuLF) is significantly higher than that of BASE and PREPP (P < 0.05). LFHFr was significantly lower than that of BASE and significantly higher than that of PREPP at PP. At POSTPP, HRVHF, HRVLF, BPVLF were significantly lower than that of BASE. But, BPVLF at POSTPP was higher than that of PP. CONCLUSIONS: We conclude that the pneumoperitoneum and trendelenburg positions caused sympathetic activation in LAVH patients.

Three-dimensional hydrodynamic-eutrophication model (HEM-3D): application to Kwang-Yang Bay, Korea.

The purpose of this paper is twofold: to describe the water quality model of Three-Dimensional Hydrodynamic-Eutrophication Model (HEM-3D) and to present an application of HEM-3D to a coastal system in Korea. HEM-3D, listed as a tool for the development of Total Maximum Daily Load by US Environmental Protection Agency, is a general-purpose modeling package for simulation of the flow field, transport, and eutrophication processes throughout the water column and of diagenetic processes in the benthic sediment. This paper describes the water quality model of HEM-3D with emphasis on its unique features. Excessive loadings of organic wastes have significantly deteriorated water quality conditions of Korean coastal waters. This paper presents an application of HEM-3D to Kwang-Yang Bay, a coastal system in Korea, which is one of the first water quality modeling efforts for Korean coastal waters accompanied by a relatively comprehensive field program. The current status of data availability for water quality modeling in Korea is discussed.