Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC. METHODS: Alteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay. RESULTS: We found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model. CONCLUSIONS: This study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC. Video Abstract.

Activity-Based Protein Profiling Reveals Potential Dasatinib Targets in Gastric Cancer.

Dasatinib is a multi-target kinase inhibitor, whose targets include BCR-ABL, SRC family kinases, and various cancer kinases. The elevated SRC activity in gastric cancer (GC) has prompted the need for the therapeutic application of dasatinib in GC. We observed that the efficacy of dasatinib varied with the GC cell lines. The differential effect of dasatinib was not correlated with the basal SRC activity of each cell line. Moreover, the GC cell lines showing the strong antitumor effects of dasatinib were refractory to other SRC inhibitors, i.e., bosutinib and saracatinib, suggesting that unexpected dasatinib's targets could exist. To profile the targets of dasatinib in GC, we performed activity-based protein profiling (ABPP) via mass spectrometry using a desthiobiotin-ATP probe. We identified 29 and 18 kinases as potential targets in dasatinib-sensitive (SNU-216, MKN-1) and -resistant (SNU-484, SNU-601) cell lines, respectively. The protein-protein interaction mapping of the differential drug targets in dasatinib-sensitive and -resistant GC using the STRING database suggested that dasatinib could target cellular energy homeostasis in the drug-sensitive GC. RNAi screening for identified targets indicated p90RSK could be a novel dasatinib target, which is important for maintaining the viability and motility of GC cells. Further functional validation of dasatinib off-target actions will provide more effective therapeutic options for GC.

Postmortem proteomics to discover biomarkers for forensic PMI estimation.

The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry-based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.

Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-associated death. However, traditional therapeutic strategies have failed to significantly improve the survival of patient with advanced GC. While KRAS mutations have been found in some patients with gastric cancer, an effective therapy to treat KRAS-driven gastric cancer has not been established yet. To provide a rationale for clinical application of kinase inhibitors targeting RAS pathways, we first determined the sensitivity of GC cell lines harboring KRAS mutations or amplification to RAS pathway inhibitors. We found that MAPK pathway inhibitors (MEKi and ERKi) were more effective than AKT inhibitor, suggesting that KRAS-driven gastric cancer cells are dependent on MAPK pathway for survival. Further, we established a KRAS mutant GC cell line with acquired resistance to MEK inhibitors in order to mimic clinical situation of kinase inhibitor resistance. A comprehensive analysis of tyrosine phosphorylation in receptor tyrosine kinases in combination with small molecule chemical library screening revealed upregulated c-MET phosphorylation in this resistance cell line with elevated sensitivity to c-MET TKI (crizotinib) and PI3K/mTOR dual inhibitor (BEZ235). We also showed that migration and invasion of resistant cells were promoted, and crizotinib and BEZ235 could inhibit this malignant phenotype. Overall, our results indicate that prolonged MAPK pathway inhibition could result in acquired resistance which is associated with increased malignant phenotype in KRAS mutant GC and pharmacological targeting c-MET and PI3K/mTOR could overcome this problem.

Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling.

Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.

Delayed Motor Development and Infant Obesity as Risk Factors for Severe Deformational Plagiocephaly: A Matched Case-Control Study.

The prevalence of deformational plagiocephaly (DP) has increased since the recommendation of positioning infants to their back during sleeping and is affected by various biological and environmental factors. This study aimed to investigate associations between DP and perinatal or infant characteristics, including obesity. This case-control study included 135 infants (81 males) aged 2-12 months who were diagnosed with DP using calculated cranial vault asymmetric index and cranial index and 135 age- and sex-matched controls. Motor development was evaluated using the Alberta Infant Motor Scale, and obesity was defined by body mass index. Univariate and multivariate logistic regression models were used to assess potential risk factors for DP and its severity. One hundred thirty-five infants with DP were divided into the following three subgroups according to severity indicated by the cranial vault asymmetry index: mild to moderate group (n = 87, 64.4%), severe group (n = 48, 35.6%), and a combined plagiocephaly and brachycephaly group (n = 79, 58.5%). Independent risk factors significantly associated with development of DP were bottle-only feeding (adjusted odds ratio (aOR) = 4.65; 95% CI: 2.70-8.00), little tummy time when awake (aOR = 3.51, 95% CI: 1.71-7.21), delay of motor development (aOR = 2.85, 95% CI: 1.08-7.49), and obesity at diagnosis (aOR = 2.45, 95% CI: 1.02-5.90). Among these risk factors, delay of motor development (aOR = 4.91, 95% CI: 1.46-16.51) and obesity at diagnosis (aOR = 4.10, 95% CI: 1.42-11.90) were particularly related to severe DP. In conclusion, this study confirms that DP risk is positively associated with bottle-only feeding, infrequent tummy time, and delayed development of motor milestones. Notably, this study demonstrates infant obesity as a new risk factor for DP. Our findings suggest that obesity should be identified early and managed comprehensively in infants with DP.

Enhanced removal of cesium by potassium-starved microalga, Desmodesmus armatus SCK, under photoheterotrophic condition with magnetic separation.

This study investigated the feasibility of using photoheterotrophic microalga, Desmodesmus armatus SCK, for removal of cesium (Cs+) followed by recovery process using magnetic nanoparticles. The comparison of three microalgae results indicated that D. armatus SCK removed the most Cs+ at both 25 °C and 10 °C. The results also revealed that the use of microalga grown in potassium (K+)-starved condition improves the accumulation of Cs+. Heterotrophic mode with addition of volatile fatty acids (VFAs), especially acetic acids (HAc), also enhanced removal of Cs+ by K+-starved D. armatus SCK; maximum removal efficiency of Cs+ was almost 2-fold higher than that of cells grown without organic carbon source. The Cs+ taken up by this microalga was efficiently harvested using magnetic nanoparticles, polydiallyldimethylammonium (PDDA)-FeO3. Finally, this strain eliminated more than 99% of radioactive 137Cs from solutions of 10, 100, and 1000 Bq mL-1. Therefore, use of K+-starved microalga, D. armatus SCK, with VFAs could be promising means to remove the Cs from the liquid wastes.

Influence of Number Fluctuation and Position Variation of Channel Dopants and Gate Metal Grains on Tunneling Field-Effect Transistors (TFETs).

The influence of number fluctuation and position variation on channel dopants and gate metal grains on tunneling field-effect transistors (TFETs) have been discussed in comparison with metal-oxide-semiconductor FETs (MOSFETs). Based on the simulation results of randomly generated device samples, the shape of the statistical threshold voltage (V(th)) distribution of TFETs associated with individual variation sources such as random dopant fluctuation (RDF) and work-function variation (WFV) have been found to be significantly different than that of MOSFETs. This analysis provides a detailed insight into the variation sources related to underlying physics of TFETs.