DDR1 promotes migration and invasion of breast cancer by modulating the Src-FAK signaling.

Breast cancer is the most commonly diagnosed cancer among women, causing 15% of patient deaths. The metastasis of breast cancer cells is the leading cause of death for patients. Several studies have shown that Discoidin Domain Receptor 1 (DDR1) was highly expressed in breast cancer and could influence tumor cell behaviors. However, the specific role of DDR1 in breast cancer metastasis is still elusive. In this study, we uncovered that DDR1 is significantly increased in breast cancer and inversely correlated with the prognosis of patients. Knockdown of DDR1 suppressed the migration and invasion of breast cancer cells. Additionally, overexpression of DDR1 enhanced the metastatic capacity of cancer cells. Immunoblotting revealed that activation of Src and FAK, which are involved in cancer cell metastasis, were correlated with the expression level of DDR1. Co-immunoprecipitation experiments showed that DDR1 could bind to Src and FAK. Finally, the inhibition of FAK and Src could attenuate DDR1 enhanced migration ability of breast cancer cells. In summary, our study revealed that DDR1 was highly expressed in breast cancer and negatively correlated with the prognosis of breast cancer patients. DDR1 facilitates migration and invasion in breast cancer cells via activation of the Src-FAK signaling. Accordingly, blocking DDR1/Src/FAK axis is a promising therapeutic strategy for breast cancer treatment.

Temozolomide promotes immune escape of GBM cells via upregulating PD-L1.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor with poor prognosis, and currently effective therapeutic strategies are still limited. Although temozolomide (TMZ) is commonly used for GBM therapy and its mechanism was well characterized, while its side effects were required comprehensive investigation. In the present study, we revealed that TMZ-challenged GBM cells strongly suppressed pro-inflammatory cytokines expression in activated periphery blood mononuclear cells (PBMC), which depended on enhanced transcription of CD274 (encoding PD-L1), but not other immune checkpoints, such as CD276, HVEM and galectin-9. Moreover, abundance of membranous PD-L1 was also increased in TMZ-treated GBM cells. When PD-L1 expression was knocked down by short hairpin RNA (shRNA), inhibitory effect of TMZ-treated GBM cells on PBMC became weakened, suggesting that PD-L1 was crucial for immune inhibition capacity of TMZ-treated GBM cells. Additionally, actinomycin D reduced PD-L1 expression in GBM cells after TMZ challenge, indicating that PD-L1 induction occurred at transcriptional level. The immunoblotting results demonstrated that STAT3 signaling was involved in TMZ-mediated PD-L1 induction, and attenuated expression of PD-L1 was observed using STAT3 inhibitor VI or STAT3 shRNA. Finally, the animal study showed that combination of TMZ and PD-1 antibody therapy strongly inhibited tumor growth and achieved the improved survival rate of GBM mice. Accordingly, this study revealed the classical chemotherapy drug TMZ promoted GBM cells immune escape, even TMZ combine with PD-1 antibody treatment not further improve survival ratio of recurrent GBM patients compared with traditional therapy methods, while our animal study provided evidence that combination of TMZ and PD-1 antibody was a promising way to treat GBM, these contradictory results indicate improving the PD-1 antibody delivery efficiency can exert strong combinational therapy outcomes.

Functional characterization of grass carp runt-related transcription factor 3: Involvement in TGF-ß1-mediated c-Myc transcription in fish cells.

In mammals, both runt-related transcription factor 3 (RUNX3) and c-Myc are the downstream effectors of transforming growth factor-ß1 (TGF-ß1) signaling to mediate various cellular responses. However, information of their interaction especially in fish is lacking. In the present study, grass carp (Ctenopharyngodon idella) runx3 (gcrunx3) cDNA was cloned and identified. Interestingly, opposing effects of recombinant grass carp TGF-ß1 (rgcTGF-ß1) on c-myc and runx3 mRNA expression were observed in grass carp periphery blood lymphocytes (PBLs). Parallelly, Runx3 protein levels were enhanced by rgcTGF-ß1 in the cells. These findings prompted us to examine whether Runx3 can mediate the inhibition of TGF-ß1 on c-myc expression in fish cells. In line with this, overexpression of grass carp Runx3 and Runx3 DN (a dominant-negative form of Runx3) in grass carp kidney cell line (CIK) cells decreased and increased c-myc transcript levels, respectively. Particularly, the regulation of Runx3 and Runx3 DN on c-myc mRNA expression was direct since they were presented in the nucleus without any stimulation. In addition, rgcTGF-ß1 alone suppressed c-myc mRNA expression in CIK cells as in PBLs. Moreover, this inhibitory effect was also observed when grass carp Runx3 and Runx3 DN were overexpressed. These results strengthened the role of TGF-ß1 signaling in controlling c-myc transcription. Taken together, TGF-ß1-mediated c-myc expression was affected at least in part by Runx3, thereby firstly exploring the functional role of Runx3 in TGF-ß1 down-regulation on c-myc mRNA expression in fish.

Reduction in activating transcription factor 4 promotes carbon tetrachloride and lipopolysaccharide/D­galactosamine­mediated liver injury in mice.

Although activating transcription factor 4 (ATF4) is involved in the regulation of numerous biological functions, whether ATF4 has a direct role in liver injury is unknown. The aim of the present study was to investigate the role of ATF4 in liver injury using mouse models. The results revealed that ATF4 protein is expressed markedly higher in the mouse liver when in comparison with other tissues. Notably, tunicamycin treatment, an endoplasmic reticulum (ER) stress inducer, induced the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), but decreased ATF4 protein levels in the mouse liver. This suggested an unconventional regulation pattern of ATF4 protein not associated with ER stress or eIF2α. In addition, it was also observed that the liver levels of ATF4 protein were significantly reduced upon chronic liver injury induced by carbon tetrachloride (CCl4). ATF4 protein was also decreased in acute liver injury induced by lipopolysaccharide (LPS) plus D­galactosamine (D­GalN). Furthermore, the results revealed that knockdown of ATF4 by injecting ATF4­targeting Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)­CRISPR associated protein 9 plasmids exacerbated CCl4 and LPS/D­GalN­induced liver injury as demonstrated by elevated serum aspartate transaminase and alanine aminotransferase levels. ATF4 suppression also enhanced CCl4 and LPS/D­GalN mediated c­Jun N­terminal kinase activation. By contrast, ATF4 overexpression alleviated CCl4 and LPS/D­GalN­induced liver injury. Taken together, these observations suggested that ATF4 may serve a protective role in the mouse liver.

Compound C induces protective autophagy in human cholangiocarcinoma cells via Akt/mTOR-independent pathway.

Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to exert antitumor activities in some types of cells. Whether compound C can exert antitumor effects in human cholangiocarcinoma (CCA) remains unknown. Here, we demonstrated that compound C is a potent inducer of cell death and autophagy in human CCA cells. Autophagy inhibitors increased the cytotoxicity of compound C towards human CCA cells, as confirmed by increased LDH release, and PARP cleavage. It is notable that compound C treatment increased phosphorylated Akt, sustained high levels of phosphorylated p70S6K, and decreased mTOR regulated p-ULK1 (ser757). Based on the data that blocking PI3K/Akt or mTOR had no apparent influence on autophagic response, we suggest that compound C induces autophagy independent of Akt/mTOR signaling in human CCA cells. Further study demonstrated that compound C inhibited the phosphorylation of JNK and its target c-Jun. Blocking JNK by SP600125 or siRNA suppressed autophagy induction upon compound C treatment. Moreover, compound C induced p38 MAPK activation, and its inhibition promoted autophagy induction via JNK activation. In addition, compound C induced p53 expression, and its inhibition attenuated compound C-induced autophagic response. Thus, compound C triggers autophagy, at least in part, via the JNK and p53 pathways in human CCA cells. In conclusion, suppresses autophagy could increase compound C sensitivity in human CCA.

c­Myc promotes cholangiocarcinoma cells to overcome contact inhibition via the mTOR pathway.

The loss of contact inhibition is a hallmark of a wide range of human cancer cells. Yet, the precise mechanism behind this process is not fully understood. c­Myc plays a pivotal role in carcinogenesis, but its involvement in regulating contact inhibition has not been explored to date. Here, we report that c­Myc plays an important role in abrogating contact inhibition in human cholangiocarcinoma (CCA) cells. Our data show that the protein level of c­Myc obviously decreased in contact-inhibited normal biliary epithelial cells. However, CCA cells sustain high protein levels of c­Myc and keep strong proliferation ability in confluent conditions. Importantly, the suppression of c­Myc by inhibitor or siRNA induced G0/G1 phase cell cycle arrest in confluent CCA cells. We demonstrate that the inhibition of c­Myc suppressed the activity of mammalian target of rapamycin (mTOR) in confluent CCA cells, and mTOR inhibition induced G0/G1 phase cell cycle arrest in confluent CCA cells. In confluent CCA cells, the activity of Merlin is downregulated, and Yes-associated protein (YAP) sustains high levels of activity. Furthermore, YAP inhibition not only induced G0/G1 phase cell cycle arrest, but also decreased c­Myc expression in confluent CCA cells. These results indicate that Merlin/YAP/c­Myc/mTOR signaling axis promotes human CCA cell proliferation by overriding contact inhibition. We propose that overriding c­Myc­mediated contact inhibition is implicated in the development of CCA.

Unfolded Protein Response Promotes Doxorubicin-Induced Nonsmall Cell Lung Cancer Cells Apoptosis via the mTOR Pathway Inhibition.

Drug resistance is extremely common in nonsmall cell lung cancer (NSCLC) and is one of the major problems in NSCLC chemotherapy. However, the detailed mechanisms remain largely unknown. Unfolded protein response (UPR) is involved in the tumorigenesis of NSCLC. Here, the authors demonstrated that the UPR promotes poly (ADP-ribose) polymerase activation (PARP) cleavage in NSCLC cells on doxorubicin treatment, which is a hallmark of apoptosis and caspase activation. In NSCLC cells, doxorubicin treatment triggers the UPR activation, which subsequently promotes doxorubicin-mediated apoptosis. Importantly, mild endoplasmic reticulum stress precondition enhances the sensitivity of NSCLC cells to doxorubicin-initiated apoptosis. Furthermore, the eukaryotic translation initiation factor 2α (eIF2α) branch of the UPR is involved in the synergistic role of the UPR in NSCLC cell apoptosis on doxorubicin treatment. They also demonstrated that the mTOR pathway plays an essential role in synergistic induction of apoptosis by the UPR and doxorubicin in NSCLC cells. Taken together, these results provide a potential mechanism that the UPR promotes doxorubicin-induced apoptosis in NSCLC cells, at least in part, by eIF2α-mediated mTOR signal inactivation.

Synergistic antitumor activity of the combination of salubrinal and rapamycin against human cholangiocarcinoma cells.

Less is known about the roles of eukaryotic initiation factor alpha (eIF2α) in cholangiocarcinoma (CCA). Here, we report that eIF2α inhibitor salubrinal inhibits the proliferation of human CCA cells. Clinical application of mammalian target of rapamycin (mTOR) inhibitors only has moderate antitumor efficacy. Therefore, combination approaches may be required for effective clinical use of mTOR inhibitors. Here, we investigated the efficacy of the combination of salubrinal and rapamycin in the treatment of CCA. Our data demonstrate a synergistic antitumor effect of the combination of salubrinal and rapamycin against CCA cells. Rapamycin significantly inhibits the proliferation of CCA cells. However, rapamycin initiates a negative feedback activation of Akt. Inhibition of Akt by salubrinal potentiates the efficacy of rapamycin both in vitro and in vivo. Additionally, rapamycin treatment results in the up-regulation of Bcl-xL in a xenograft mouse model. It is notable that salubrinal inhibits rapamycin-induced Bcl-xL up-regulation in vivo. Taken together, our data suggest that salubrinal and rapamycin combination might be a new and effective strategy for the treatment of CCA.

Molecular and functional characterization of an IL-1ß receptor antagonist in grass carp (Ctenopharyngodon idella).

In the present study, we discovered a novel IL-1 family member (nIL-1F) from grass carp that possessed the ability to bind with grass carp IL-1ß receptor type 1 (gcIL-1R1) and attenuate grass carp IL-1ß activity in head kidney leukocytes (HKLs), suggesting that it may function as an IL-1ß receptor antagonist. Grass carp nIL-1F transcript was constitutively expressed with the highest levels in some lymphoid organs, including head kidney, spleen and intestine, implying its potential in grass carp immunity. In agreement with this notion, in vitro and in vivo studies showed that nIL-1F mRNA was inductively expressed in grass carp with a rapid kinetics, indicating that it may be an early response gene during immune challenges. In addition, recombinant grass carp IL-1ß (rgcIL-1ß) induced nIL-1F mRNA expression via NF-κB and MAPK (JNK, p38 and p42/44) signaling pathways in HKLs. Particularly, the orthologs of nIL-1F found in other fish species, including zebrafish, pufferfish and rainbow trout are not homologous to mammalian IL-1ß receptor antagonist (IL-1Ra), indicating that fish nIL-1F and mammalian IL-1Ra may not share a common evolutionary ancestor. Taken together, our data suggest the existence of a naturally occurring fish nIL-1F, which may function like mammalian IL-1Ra, being beneficial to understand the auto-regulatory mechanism of IL-1ß activity in fish immunity.

Molecular identification of transcription factor Runx1 variants in grass carp (Ctenopharyngodon idella) and their responses to immune stimuli.

The Runt-related transcription factor (Runx) family consists of three members, Runx1, Runx2 and Runx3 in mammals, which are involved in various biological processes. Recent studies have demonstrated that Runx1 plays critical roles in the immunity of higher vertebrates. In fish, zebrafish and fugu Runx family members have been identified, and their chromosome location, promoter usage and expression patterns have been elucidated. However, their expression profiles in immune responses are still unknown. In this study, we identified grass carp five Runx1 (gcRunx1) variants (v1-5) possibly generated through alternative promoter usage and alternative splicing. The gcRunx1 v1-3 encodes the proteins possessing intact structural characteristics of Runx family, but the putative proteins of gcRunx1 v4-5 lack a transactivation domain, an inhibitory domain and a C-terminal pentapeptide motif (VWRPY). Tissue distribution assays revealed that gcRunx1 was preferentially expressed in some immune-related tissues including thymus and spleen, indicating its potential roles in teleost immunity. The changes of gcRunx1 expression to various immune stimuli was examined in periphery blood lymphocytes, showing that gcRunx1 v1-3 mRNA levels were increased after LPS, poly I:C and PHA treatment, whereas gcRunx1 v4-5 mRNA expression were stimulated only by LPS and PHA. Furthermore, in vivo studies confirmed that bacterial challenge enhanced gcRunx1 mRNA levels. In particular, in vitro and in vivo studies revealed that gcRunx1 v4-5 mRNA expression was induced with a delayed kinetics compared with that of gcRunx1 v1-3. These findings not only provide the evidence for the involvement of gcRunx1 in immune response, but also reveal the inducible expression diversity of fish Runx1 splicing variants, thereby facilitating further elucidating the role of Runx1 in piscine immunity.

[Identification of phosphoproteome in mice neurons by isotope-labeling technique combining with chemiluminescence Western blotting method].

This study was aimed to work out a simple, applicable, sensitive and specific protocol for the identification of phosphoproteome. Isotope-labeling, two-dimensional electrophoresis, autoradiography and so on were used to establish a phosphoproteome map of mice neurons, and then chemiluminescence Western blotting was utilized to detect three phosphoproteins PI3Kr3, MEK1 and PKCalpha selectively. The results of comparison showed that the blots of PI3Kr3, MEK1 and PKCalpha on autoradiography map were almost identical with the blots of PI3Kr3, MEK1 and PKCalpha on chemiluminescence Western blotting maps. So this protocol based on isotope labeling and chemiluminescence Western blotting methods has proven to be sensitive and specific in the identification of phosphoproteome.

[Morphological changes of non-apoptotic programmed cell death of polymorphonuclar neutrophils induced by ONO-AE-248].

AIM: To investigate the morphological changes of polymorphonuclar neutrophils (PMNs) induced by ONO-AE-248, a selective agonist of EP3. METHODS: The morphological changes of PMNs treated with or without ONO-AE-248 were observed by electron microscope, fluorescence microscope and confocal microscope. RESULTS: A unique form of PMNs death was rapidly caused by ONO-AE-248. The agonist primarily induced morphological changes of PMN nucleus under electron microscope, including the fusion of the nuclear lobules same as a large round structure, decreasing of the compactness of chromatin, the blebbing and rupture of nuclear membrance. Observation of PMN nucleus under fluorescence microscope by DAPI staining gave the same conclusion. However, there were no apparent changes in other intracellular organelles. The structure and distribution of mitochondria of PMNs treated with ONO-AE-248 were different from the typical morphological changes of apoptotic PMNs. ONO-AE-248 exerted little effects on the exposure of cell membrane phosphatidylserine (PS). CONCLUSION: ONO-AE-248 can promote the non-apoptotic programmed cell death of PMNs in vitro. The events occurring in nucleus and mitochondria might be the early features of the novel cell death, which suggests that nucleus and mitochondria may be the reaction center of PMNs induced by ONO-AE-248.