Stromal Rigidity Stress Accelerates Pancreatic Intraepithelial Neoplasia Progression and Chromosomal Instability via Nuclear Protein Tyrosine Kinase 2 Localization.

Because the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and phosphorylated Ser-139 residue of the histone variant H2AX (γH2AX) foci linked to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3-µm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell data sets revealed that the extracellular matrix shaped an alternate route of acinar-ductal transdifferentiation of acinar cells into topoisomerase II α (TOP2A)-overexpressing cancer cells and derived subclusters with copy number amplifications in MYC-PTK2 (protein tyrosine kinase 2) locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in The Cancer Genome Atlas-Pancreatic Adenocarcinoma cohort. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased vav guanine nucleotide exchange factor 1 (VAV1) expression, and prolonged overall survival in the KPC mice. Reduction of α-smooth muscle actin deposition in the CD248 knockout KPC mice remodeled the tissue stroma and down-regulated TOP2A expression in the epithelium. In summary, stromal stiffness induced the onset of cancer cells-of-origin by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment.

RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m5C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m5C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m5C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-KrasG12D/+;Trp53fl/fl;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant KrasG12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. SIGNIFICANCE: Transcriptome-wide mRNA bisulfite sequencing identified candidate m5C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m5C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.

Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

GIPR agonism mediates weight-independent insulin sensitization by tirzepatide in obese mice.

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Epigenetic silencing of AATK in acinar to ductal metaplasia in murine model of pancreatic cancer.

BACKGROUND: Cancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA). RESULTS: By analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice. CONCLUSION: Epigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

A colorimetric detection of microRNA-148a in gastric cancer by gold nanoparticle-RNA conjugates.

For the early diagnosis of gastric cancer, microRNA-148a (miRNA-148a) as a promising biomarker is measured by a simple colorimetric biosensor due to its unique surface plasmon resonance (SPR) absorption of gold nanoparticles (AuNPs). In the assay system, the sensing probes are facilitated by the conjugation of AuNPs with RNA probes (RNAP) via Au-S bonds, which align in a tail-to-tail fashion onto the target RNA. When miRNA-148a is introduced, a sandwich hybridization reaction is triggered between the AuNP-RNAP conjugates and targets, resulting in changes in the SPR absorption band, microscopic distribution and macroscopic color of the AuNP solution. Following this principle, this colorimetric method is able to quantitatively detect miRNA-148a at nanomolar level with a limit of ∼1.9 nM, and exhibits high sensitivity and selectivity by a low-cost UV-vis spectrometer or even the naked eye. Moreover, the AuNP network materials with a characteristic sharp 'melting transition' provide significant guidance for the reusability of DNA or RNA biosensors.

Ultrasensitive NO Gas Sensor Based on the Graphene Oxide-Coated Long-Period Fiber Grating.

An ultrasensitive nitric oxide (NO) gas sensor based on the graphene oxide (GO)-coated long-period fiber grating (LPFG) was constructed successfully because of its excellent sensitivity to the surrounding refractive index (SRI) change. The surface morphology and structure of GO coated on LPFG were characterized by the scanning electron microscope (SEM), scanning probe microscope (SPM), and Raman spectroscopy, respectively. The adsorption principle of NO molecules by GO was calculated in detail by density functional theory (DFT) and further characterized by Fourier transform infrared spectrometry (FT-TR) and X-ray photoelectron spectroscopy (XPS). Our studies demonstrate that the adsorption principle of NO molecules by GO was the combined effect of physical adsorption and chemical adsorption because of the formation of C-N bonds between GO and NO and the oxidization of NO to NO2. The NO sensor exhibits excellent sensing performance in the NO concentration range of 0 to 400 ppm.

Applications of carbon quantum dots to alleviate Cd2+ phytotoxicity in Citrus maxima seedlings.

Heavy metal pollution may affect plant growth. The focus of this study was to explore remediation agents that alleviate cadmium toxicity in plants. The Citrus maxima (grapefruit) seedlings were cultivated for 10 days under hydroponic conditions amended with different concentrations of Cd2+ (50 and 200 mg/L) and CDs (600 and 900 mg/L). Our observations on roots and leaves showed that, the plant exposed to 200 mg/L Cd2+ alone was damaged, supported by the changes in anthocyanin contents, activity of antioxidant enzymes and cell membrane peroxidation damage (up to 35.8-45%). However, the physiological properties of the plant were improved upon exposed to 200 mg/L Cd2+ plus 900 mg/L CDs; it can be ascribed to Cd2+ sorption to the co-exposed CDs which reduced its freely dissolved concentration by more than 22.5%, thus significantly reducing the amount of Cd2+ entered the plant roots by 50.7-89.4%. Due to the oxidative stress induced by Cd2+ exposure at 200 mg/L, expression of glutathione-producing genes was up-regulated by 30-360% relative to the control, while the genes expression upon exposure to 200 mg/L Cd2+ and 900 mg/L CDs was reduced by 48.4-91.5% relative to that exposed to 200 mg/L Cd2+ alone. However, detoxification of CDs on plant leaves at 600 mg/L was insignificant, because a portion of Cd2+ taken up by roots can be transported to leaves associated with the internalized CDs. Therefore, CDs can be utilized as a repair agent to mitigate toxicity of Cd2+ to plant especially at a high amendment level (900 mg/L).

p21-Activated kinase 3 promotes cancer stem cell phenotypes through activating the Akt-GSK3ß-ß-catenin signaling pathway in pancreatic cancer cells.

Relative to several other p21-activated kinase (PAK) family members, the role of PAK3 in regulating cancer cell functions remains unclear. Our study obtained evidence that PAK3 regulates the Akt-GSK3ß-ß-catenin signaling by acting as Ser473-Akt kinase in several pancreatic cancer cell lines. Specifically, knockdown of PAK3 or overexpression of dominant-negative PAK3 inhibited the phosphorylation of Ser473-Akt and GSK3ß, resulting in the proteasomal degradation of ß-catenin. Conversely, overexpression of PAK3 led to activation of Akt signaling and increased ß-catenin expression. These changes, however, were not noted with the silencing and/or overexpression of PAK1, PAK2, or PAK4, which underlies the impetus of PAK3 as a key effector in governing malignant phenotypes in these pancreatic cancer cells, including cancer stem cell (CSC) expansion. Accordingly, PAK3 depletion effectively suppresses tumorsphere formation, ALDH activity, and the expression of CSC surface markers. Moreover, we used a stable knockdown clone of AsPC-1 cells to demonstrate the in vivo efficacy of PAK3 inhibition in suppressing tumorigenesis and xenograft tumor growth. Together, these findings suggest the potential role of PAK3 as a target for pancreatic cancer therapy, which warrants further investigations.

Suppression of Tumor Growth and Muscle Wasting in a Transgenic Mouse Model of Pancreatic Cancer by the Novel Histone Deacetylase Inhibitor AR-42.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. This study was aimed at evaluating the efficacy of AR-42 (formerly OSU-HDAC42), a novel histone deacetylase (HDAC) inhibitor currently in clinical trials, in suppressing tumor growth and/or cancer-induced muscle wasting in murine models of PDAC. EXPERIMENTAL DESIGN: The in vitro antiproliferative activity of AR-42 was evaluated in six human pancreatic cancer cell lines (AsPC-1, COLO-357, PANC-1, MiaPaCa-2, BxPC-3, SW1990). AsPC-1 subcutaneous xenograft and transgenic KPfl/flC (LSL-KrasG12D;Trp53flox/flox;Pdx-1-Cre) mouse models of pancreatic cancer were used to evaluate the in vivo efficacy of AR-42 in suppressing tumor growth and/or muscle wasting. RESULTS: Growth suppression in AR-42-treated cells was observed in all six human pancreatic cancer cell lines with dose-dependent modulation of proliferation and apoptotic markers, which was associated with the hallmark features of HDAC inhibition, including p21 upregulation and histone H3 hyperacetylation. Oral administration of AR-42 at 50 mg/kg every other day resulted in suppression of tumor burden in the AsPC-1 xenograft and KPfl/flC models by 78% and 55%, respectively, at the end of treatment. Tumor suppression was associated with HDAC inhibition, increased apoptosis, and inhibition of proliferation. Additionally, AR-42 as a single agent preserved muscle size and increased grip strength in KPfl/flC mice. Finally, the combination of AR-42 and gemcitabine in transgenic mice demonstrated a significant increase in survival than either agent alone. CONCLUSIONS: These results suggest that AR-42 represents a therapeutically promising strategy for the treatment of pancreatic cancer.

Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH.

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.