Deacetylation of ß-catenin by SIRT1 regulates self-renewal and oncogenesis of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is a highly malignant liver tumor. The presence of cancer stem cells (CSCs) figures prominently in tumor invasion, therapeutic resistance and tumor recurrence resulting in poor outcome and limited therapeutic options. Wnt/ß-catenin signaling is essential for cancer stem cell regulation and tumorigenesis in HCC, but its molecular mechanisms are not fully understood. Here, we demonstrate that ß-catenin is overexpressed in liver CSCs, and its expression level is positively correlated with SIRT1 in HCC specimens. SIRT1 regulates the protein stability of ß-catenin, thereby affecting the transcriptional activity of Wnt/ß-catenin signaling in liver CSCs. Mechanistically, we show that nuclear accumulation of ß-catenin results from deacetylation mediated by SIRT1. Further, nuclear ß-catenin promotes the transcription of Nanog to help maintain self-renewal of liver CSCs. Taken together, our findings indicate that the deacetylation of ß-catenin by SIRT1 represents a critical mechanism for regulating liver CSCs self-renewal and tumorigenesis. It provides an improved understanding of molecular mechanisms underlying ß-catenin activation and tumorigenesis in HCC.

Association Between Activation of the Programmed Cell Death-1 (PD-1)/Programmed Death-Ligand 1 (PD-L1) Pathway and Pain in Patients with Cancer.

BACKGROUND The aim of this study was to investigate the clinical correlation between sPD-1 (soluble programmed cell death-1) and PD-1 (programmed cell death-1) expression and cancer pain. MATERIAL AND METHODS sPD-1 content in peripheral blood was determined by enzyme-linked immunosorbent assay (ELISA). T cell surface-positive rate was determined by flow cytometry, and the correlation of clinical characteristics of patients with cancer pain was analyzed. RESULTS The positive expression rate of PD-1 in sPD-1 and T cells of patients with cancer pain was higher than that in normal patients. There was a significant correlation between sPD-1 and PD-1 positivity on T cell surface with tumor type, differentiation degree, and VAS scores of patients with cancer pain (P<0.05). Peripheral blood sPD-1 level and PD-1 positivity in patients with liver cancer and melanoma cancer were higher than those in patients with renal cell carcinoma and breast cancer. In addition, peripheral blood sPD-1 level and PD-1 positivity in patients with poorly-differentiated cancer pain were higher than those in patients with intermediately- to well-differentiated cancer. The sPD-1 content was lower and PD-1 positivity rate was higher in cancer pain patients with low VAS scores. CONCLUSIONS The positive expression rate of sPD-1 and PD-1 in patients with cancer pain is higher than that in normal people. The activation rate of the PD-1/PD-L1 pathway was mediated by sPD-1 and PD-1 positive expression, age, tumor type, and differentiation. There are correlations between clinical characteristics such as degree and pain level as shown by VAS score.

Tissue expression pattern of ABCG transporter indicates functional roles in reproduction of Toxocara canis.

Toxocara canis is a zoonotic parasite with worldwide distribution. ATP-binding cassette (ABC) transporters are integral membrane proteins which involve in a range of biological processes in various organisms. In present study, the full-length coding sequence of abcg-5 gene of T. canis (Tc-abcg-5) was cloned and characterized. A 633 aa polypeptide containing two conserved Walker A and Walker B motifs was predicted from a continuous 1902 nt open reading frame. Quantitative real-time PCR was employed to determine the transcriptional levels of Tc-abcg-5 gene in adult male and female worms, which indicated high mRNA level of Tc-abcg-5 in the reproductive tract of adult female T. canis. Tc-abcg-5 was expressed to produce rabbit polyclonal antiserum against recombinant TcABCG5. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of TcABCG5, which showed predominant distribution of TcABCG5 in the uterus (especially in the germ cells) of adult female T. canis. Tissue transcription and expression pattern of Tc-abcg-5 indicated that Tc-abcg-5 might play essential roles in the reproduction of this parasitic nematode.

Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers.

Chimeric antigen receptor T (CAR-T) cells have shown promising efficacy in treatment of hematological malignancies, but its applications in solid tumors need further exploration. In this study, we investigated CAR-T therapy targeting carcino-embryonic antigen (CEA)-positive colorectal cancer (CRC) patients with metastases to evaluate its safety and efficacy. Five escalating dose levels (DLs) (1 × 105 to 1 × 108/CAR+/kg cells) of CAR-T were applied in 10 CRC patients. Our data showed that severe adverse events related to CAR-T therapy were not observed. Of the 10 patients, 7 patients who experienced progressive disease (PD) in previous treatments had stable disease after CAR-T therapy. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level was apparent in most patients even in long-term observation. Furthermore, we observed persistence of CAR-T cells in peripheral blood of patients receiving high doses of CAR-T therapy. Importantly, we observed CAR-T cell proliferation especially in patients after a second CAR-T therapy. Taken together, we demonstrated that CEA CAR-T cell therapy was well tolerated in CEA+ CRC patients even in high doses, and some efficacy was observed in most of the treated patients.

Involvement of ethylene and polyamines biosynthesis and abdominal phloem tissues characters of wheat caryopsis during grain filling under stress conditions.

Severe water deficit (SD) severely limited the photo-assimilate supply during the grain-filling stages. Although the ethylene and polyamines (PAs) have been identified as important signaling molecules involved in stress tolerance, it is yet unclear how 1-Aminocylopropane-1-carboxylic acid (ACC) and PA biosynthesis involving wheat abdominal phloem characters mitigate SD-induced filling inhibition. The results obtained indicated that the SD down-regulated the TaSUT1 expression and decreased the activities of sucrose synthase (SuSase, EC2.4.1.13), ADP glucose pyrophosphorylase (AGPase, EC2.7.7.27), soluble starch synthase (SSSase, EC2.4.1.21), then substantially limited grain filling. As a result, increased ACC and putrescine (Put) concentrations and their biosynthesis-related gene expression reduced spermidine (Spd) biosynthesis under SD condition. And, the ACC and PA biosynthesis in inferior grains was more sensitive to SD than that in superior grains. Intermediary cells (ICs) of caryopsis emerged prematurely under SD to compensate for the weakened photo-assimilate transport functions of sieve elements (SEs). Finally, plasmolysis and nuclear chromatin condensation of phloem parenchyma cells (PPC) and membrane degradation of SEs, as well as the decreased ATPase activity on plasma membranes of ICs and PPC at the later filling stage under SD were responsible for the considerably decreased weight of inferior grains.

MEF2D Transduces Microenvironment Stimuli to ZEB1 to Promote Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) is an essential mechanism of metastasis, including in colorectal cancer. Although EMT processes are often triggered in cancer cells by their surrounding microenvironment, how EMT-relevant genes control these processes is not well understood. In multiple types of cancers, the transcription factor MEF2D has been implicated in cell proliferation, but its contributions to metastasis have not been addressed. Here, we show MEF2D is overexpressed in clinical colorectal cancer tissues where its high expression correlates with metastatic process. Functional investigations showed that MEF2D promoted cancer cell invasion and EMT and that it was essential for certain microenvironment signals to induce EMT and metastasis in vivo Mechanistically, MEF2D directly regulated transcription of the EMT driver gene ZEB1 and facilitated histone acetylation at the ZEB1 promoter. More importantly, MEF2D responded to various tumor microenvironment signals and acted as a central integrator transducing multiple signals to activate ZEB1 transcription. Overall, our results define a critical function for MEF2D in upregulating EMT and the metastatic capacity of colorectal cancer cells. Further, they offer new insights into how microenvironment signals activate EMT-relevant genes and deepen the pathophysiologic significance of MEF2D, with potential implications for the prevention and treatment of metastatic colorectal cancer. Cancer Res; 76(17); 5054-67. ©2016 AACR.

Exogenous Cytokinins Increase Grain Yield of Winter Wheat Cultivars by Improving Stay-Green Characteristics under Heat Stress.

Stay-green, a key trait of wheat, can not only increase the yield of wheat but also its resistance to heat stress during active photosynthesis. Cytokinins are the most potent general coordinator between the stay-green trait and senescence. The objectives of the present study were to identify and assess the effects of cytokinins on the photosynthetic organ and heat resistance in wheat. Two winter wheat cultivars, Wennong 6 (a stay-green cultivar) and Jimai 20 (a control cultivar), were subjected to heat stress treatment from 1 to 5 days after anthesis (DAA). The two cultivars were sprayed daily with 10 mg L-1 of 6-benzylaminopurine (6-BA) between 1 and 3 DAA under ambient and elevated temperature conditions. We found that the heat stress significantly decreased the number of kernels per spike and the grain yield (P < 0.05). Heat stress also decreased the zeatin riboside (ZR) content, but increased the gibberellin (GA3), indole-3-acetic acid (IAA), and abscisic acid (ABA) contents at 3 to 15 DAA. Application of 6-BA significantly (P < 0.05) increased the grain-filling rate, endosperm cell division rate, endosperm cell number, and 1,000-grain weight under heated condition. 6-BA application increased ZR and IAA contents at 3 to 28 DAA, but decreased GA3 and ABA contents. The contents of ZR, ABA, and IAA in kernels were positively and significantly correlated with the grain-filling rate (P < 0.05), whereas GA3 was counter-productive at 3 to 15 DAA. These results suggest that the decrease in grain yield under heat stress was due to a lower ZR content and a higher GA3 content compared to that at elevated temperature during the early development of the kernels, which resulted in less kernel number and lower grain-filling rate. The results also provide essential information for further utilization of the cytokinin substances in the cultivation of heat-resistant wheat.

Non-CSCs nourish CSCs through interleukin-17E-mediated activation of NF-κB and JAK/STAT3 signaling in human hepatocellular carcinoma.

Within the cancer stem cell (CSC) niche, non-CSCs play an indispensable role in facilitating a microenvironment capable of maintaining CSC properties. Non-CSCs contribute to not only the structure and topology of the tumor microenvironment but also the maintenance of the dynamic state of CSCs. Interleukin-17E (IL-17E/IL-25) is important in allergic inflammation and protection against parasitic infection. Moreover, it has also been demonstrated that IL-17E takes part in different cancers recently. Here, for the first time we demonstrate that discrepant expression of IL-17E and the IL-17 receptor B (IL-17RB) exists in Nanog positive (Nanog(Pos)) CSCs and Nanog negative (Nanog(Neg)) non-CSCs in hepatocellular carcinoma (HCC). Moreover, we further demonstrate that IL-17E binding to IL-17RB activates NF-κB and JAK/Stat3 pathways to promote proliferation and sustain self-renewal of CSCs in HCC. Meanwhile, the beneficial effect of IL-17E on Nanog(Pos) CSCs could be blocked by specific inhibitors of JAK and NF-κB signaling. All the findings indicated that non-CSC-derived secreted IL-17E binds IL-17RB on CSCs to signal via JAK/Stat3 and NF-κB pathways to mediate crosstalk between CSCs and non-CSCs. Therefore, IL-17E/IL-17RB signaling represents a potential therapeutic target for treatment of HCC.

SIRT1 promotes epithelial-mesenchymal transition and metastasis in colorectal cancer by regulating Fra-1 expression.

Understanding molecular mechanisms of colorectal cancer (CRC) metastasis is urgently required for targeted therapy and prognosis of metastatic CRC. In this study, we explored potential effects of silent mating type information regulation 2 homolog 1 (SIRT1) on CRC metastasis. Our data showed that ectopic expression of SIRT1 markedly increased the migration and invasion of CRC cells. In contrast, silencing SIRT1 repressed this behavior in aggressive CRC cells. Tumor xenograft experiments revealed that knockdown of SIRT1 impaired CRC metastasis in vivo. Silencing SIRT1 in CRC cells induced mesenchymal-epithelial transition (MET), which is the reverse process of epithelial-mesenchymal transition (EMT) and characterized by a gain of epithelial and loss of mesenchymal markers. We provided a mechanistic insight toward regulation of Fra-1 by SIRT1 and demonstrated a direct link between the SIRT1-Fra-1 axis and EMT. Moreover, SIRT1 expression correlated positively with Fra-1 expression, metastasis and overall survival in patients with CRC. Taken together, our data provide a novel mechanistic role of SIRT1 in CRC metastasis, suggesting that SIRT1 may serve as a potential therapeutic target for metastatic CRC.

Gastrin attenuates ischemia-reperfusion-induced intestinal injury in rats.

Intestinal ischemia-reperfusion (I/R) injury is a devastating complication when the blood supply is reflowed in ischemic organs. Gastrin has critical function in regulating acid secretion, proliferation, and differentiation in the gastric mucosa. We aimed to determine whether gastrin has an effect on intestinal I/R damage. Intestinal I/R injury was induced by 60-min occlusion of the superior mesenteric artery followed by 60-min reperfusion, and the rats were induced to be hypergastrinemic by pretreated with omeprazole or directly injected with gastrin. Some hypergastrinemic rats were injected with cholecystokinin-2 (CCK-2) receptor antagonist prior to I/R operation. After the animal surgery, the intestine was collected for histological analysis. Isolated intestinal epithelial cells or crypts were harvested for RNA and protein analysis. CCK-2 receptor expression, intestinal mucosal damage, cell apoptosis, and apoptotic protein caspase-3 activity were measured. We found that high gastrin in serum significantly reduced intestinal hemorrhage, alleviated extensive epithelial disruption, decreased disintegration of lamina propria, downregulated myeloperoxidase activity, tumor necrosis factor-α, and caspase-3 activity, and lead to low mortality in response to I/R injury. On the contrary, CCK-2 receptor antagonist L365260 could markedly impair intestinal protection by gastrin on intestinal I/R. Severe edema of mucosal villi with severe intestinal crypt injury and numerous intestinal villi disintegrated were observed again in the hypergastrinemic rats with L365260. The survival in the hypergastrinemic rats after intestinal I/R injury was shortened by L365260. Finally, gastrin could remarkably upregulated intestinal CCK-2 receptor expression. Our data suggest that gastrin by omeprazole remarkably attenuated I/R induced intestinal injury by enhancing CCK-2 receptor expression and gastrin could be a potential mitigator for intestinal I/R damage in the clinical setting.

MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways.

Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF-1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVP-AEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance.