SPOP mutations promote tumor immune escape in endometrial cancer via the IRF1-PD-L1 axis.

Blockade of programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L1) has evolved into one of the most promising immunotherapy strategies for cancer patients. Tumor cells frequently overexpress PD-L1 to evade T cell-mediated immune surveillance. However, the specific genetic alterations that drive aberrant overexpression of PD-L1 in cancer cells remain poorly understood. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in endometrial cancer (EC). Here, we report that SPOP negatively regulates PD-L1 expression at the transcriptional level. Wild-type SPOP binds to IRF1, a primary transcription factor responsible for the inducible expression of PD-L1, and subsequently triggers its ubiquitin- proteasomal degradation to suppress IRF1-mediated transcriptional upregulation of PD-L1. In contrast, EC-associated SPOP mutants lose their capacity to degrade IRF1 but stabilize IRF1, and upregulate PD-L1 expression. EC-associated SPOP mutations accelerate xenograft tumor growth partially by increasing IRF1 and PD-L1 expression. Together, we identify SPOP as a negative regulator of the IRF1-PD-L1 axis and characterize the critical roles of IRF1 and PD-L1 in SPOP mutation-driven tumor immune evasion in EC.

SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate cancer.

p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.

Novel self-nanomicellizing formulation based on Rebaudioside A: A potential nanoplatform for oral delivery of naringenin.

In the study described here, we strove to develop an orally administered novel self-nanomicellizing formulation based on Rebaudioside A (RA) for delivering naringenin (NAR) with improved bioavailability and therapeutic efficacy. Our research found that RA and naringenin (NAR) could be formulated into self-assembling nanomicelles (RA-NAR) using a simple ethanol dissolution-evaporation method. We found that the RA-NAR self-assemblies comprised ultra-small micelles (5.234 ± 0.311 nm) in a uniform dispersion state (the polydispersity index was 0.243 ± 0.039) with a near-neutral surface charge (-[2.268 ± 0.729] mV). We also found that RA-NAR had a well-storage stability at 4 °C with light protection. In addition, we observed that RA-NAR exhibited enhanced apparent solubility, in-vitro permeability, and antioxidant activity. After we administered RA-NAR to rats orally, we observed an increase in area under the curve (AUC0→t) to 19,500.82 ng/mL/h versus 9324.47 ng/mL/h observed with free NAR and an increase of maximum concentration (Cmax) to 27,326.10 ng/mL from the free-NAR Cmax level of 2549.04 ng/mL. The tissue distribution assessments further demonstrated that RA-NAR could effectively increase the NAR concentration in all tested intestinal segments. Our mouse model results showed as well that oral administration of RA-NAR could efficiently protect against small intestine injuries induced by indomethacin, and the mechanisms by inhibiting proinflammatory cytokines and oxidative stress were involved in its therapeutic effect. Taken together, these findings indicate that a self-nanomicellizing formulation based on RA has great potential as a novel oral nano-drug delivery system for NAR.

Vinculin promotes gastric cancer proliferation and migration and predicts poor prognosis in patients with gastric cancer.

Vinculin is a highly conserved protein involved in cell proliferation, migration, and adhesion. However, the effects of vinculin on gastric cancer (GC) remain unclear. Therefore, we aimed to explore the functional role of vinculin in GC, as well as its underlying mechanism. Expression of vinculin in patients with GC was analyzed by real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. Overall survival was evaluated by the Kaplan-Meier method with the log-rank test. The relationship between vinculin and clinicopathological characteristics of patients with GC was further identified. In addition, we assessed the expression of vinculin in GC cell lines. Besides, vinculin was suppressed or overexpressed by transfection with small interfering (si-vinculin) or pcDNA-vinculin and then cell viability, cell apoptosis, and/or migration was respectively examined by the 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide assay, flow cytometer, and scratch assay, respectively. Moreover, the cell cycle- and apoptosis-related proteins were detected by Western blot analysis. The expression of vinculin was significantly increased in the GC tissues and cells compared with the nontumor tissues or cells. Vinculin protein positive staining was mainly located in the cell membrane and cytoplasm. Moreover, vinculin was significantly associated with Tumor Node Metastasis (TNM) and poor differentiation. Patients with high vinculin levels had significantly worse overall survival than those with low levels. Suppression of vinculin significantly decreased cell viability and migration and promoted cell apoptosis. However, overexpression of vinculin statistically increased cell viability but had no effects on cell apoptosis. Vinculin promotes GC proliferation and migration and predicts poor prognosis in patients with GC.

miR-129-3p inhibits NHEJ pathway by targeting SAE1 and represses gastric cancer progression.

Gastric cancer (GC) is one of the leading malignancies worldwide and is also a leading cause of cancer-related mortality. Micro RNA (miRNA) is a group of short non-coding RNAs modulating gene expression through targeting the 3'UTR of genes. Peripheral blood exosome miRNAs are relatively stable and might be used as biomarkers for clinical diagnosis. In this study, we found a reduced level of miR-129-3p in GC tumor when compared with adjacent non-tumor tissues, and the peripheral blood exosome miR-129-3p level was also reduced when compared with healthy controls. Subsequently, we identified that miR-129-3p repressed the expression of SUMO-activating enzyme subunit 1 (SAE1) directly through targeting 3'UTR. miR-129-3p also inhibited the sumoylation modification of XRCC4 which disturbed the nuclear localization of XRCC4 and induced more DNA damage in GC cells. Furthermore, overexpression of miR-129-3p induced more cell apoptosis and inhibited GC cell proliferation, migration and invasion, indicating miR-129-3p is a powerful anti-tumor miRNA and has potential for GC treatment.

5-Azacytidine suppresses EC9706 cell proliferation and metastasis by upregulating the expression of SOX17 and CDH1.

5-Azacytidine is a well-known anticancer drug that is clinically used in the treatment of breast cancer, melanoma and colon cancer. It has been reported that 5-azacytidine suppresses the biological behavior of esophageal cancer cells. However, corresponding mechanisms remain unclear. In this study, using Transwell invasion and cell proliferation assays, we demonstrated that 5-azacytidine significantly inhibited the metastasis and proliferation of EC9706 cells, and upregulated the expression of cadherin 1 (CDH1) and SRY-box containing gene 17 (SOX17). Moreover, the inhibition of the metastasis of the 5-azacytidine-treated EC9706 cells was impaired following transfection with siRNA targeting CDH1 (CDH1 siRNA), and the inhibition of cell proliferation was attenuated following the downregulation of SOX17 by siRNA targeting SOX17 (SOX17 siRNA). Furthermore, 5-azacytidine remarkably reduced the CDH1 and SOX17 promoter methylation levels, suggesting that 5-azacytidine upregulates the expression of SOX17 and CDH1 by inhibiting the methylation of the SOX17 and CDH1 promoter. The findings of our study confirm that 5-azacytidine suppresses the proliferation and metastasis of EC9706 esophageal cancer cells by upregulating the expression of CDH1 and SOX17. The expression levels of CDH1 and SOX17 negatively correlate with the promoter methylation levels. CDH1 and SOX17 are potential indicators of the clinical application of 5-azacytidine.

Neuropilin-1 is associated with clinicopathology of gastric cancer and contributes to cell proliferation and migration as multifunctional co-receptors.

BACKGROUND: Neuropilin-1 (NRP-1) is a transmembrane glycoprotein participating in the growth and metastasis of cancer cells as multifunctional co-receptors by interacting with the signaling pathways. However, its role in gastric cancer has not yet been clarified. This study aims to investigate whether NRP-1 expression is associated with the clinicopathology of gastric cancer, and involved in the growth and metastasis of gastric cancer cells. METHODS: NRP-1 expression in clinical gastric cancer specimens was examined by immunohistochemistry and its association with clinicopathology analyzed. The expression of NRP-1 in a panel of human gastric cancer cells was examined by real-time RT-PCR and immunoblotting. Stable transfectants depleted of NRP-1, termed MGC-803-NRP(low), were generated from MGC-803 cells. Cell proliferation was analyzed by the Cell Counting Kit-8 and Bromodeoxyuridine incorporation assays, and migrating ability analyzed by migration assays. The xenograft model was used to assess the effects of NRP-1 depletion on tumorigenesis, growth, metastasis and therapeutic potentials. The role of NRP-1 as co-receptors in the signaling pathways stimulated by ligands was examined. The key molecules involved in cell proliferation, migration and related signaling pathways were detected by immunoblotting. RESULTS: Gastric cancer tissues expressed higher levels of NRP-1 compared to normal gastric mucosa. Its expression correlated with clinical staging, tumor differentiation and pathological types. NRP-1 depletion inhibited cell proliferation by inducing cell cycle arrest in the G1/S phase by upregulating p27, and downregulating cyclin E and cyclin-dependent kinase 2. NRP-1 depletion reduced the ability of cells to migrate by inhibiting the phosphorylation of focal adhesion kinase. NRP-1 depletion suppressed tumorigenesis, tumor growth and lung metastasis by inhibiting cell proliferation and tumor angiogenesis in situ. Therapeutic NRP-1 shRNA inhibited the growth of established BGC823 tumors. Depletion of NRP-1 inhibited the activation of VEGF/VEGFR2, EGF/EGFR and HGF/c-Met pathways stimulated by respective recombinant human VEGF-165, EGF and HGF proteins. CONCLUSIONS: The present results indicate that NRP-1 may be a potentially valuable biomarker and therapeutic target for gastric cancer.

Novel impurity-free hexagonal hydroxyapatite nanotubes for local delivery of antibiotics in orthopedic surgery: in vitro release validation.

Hydroxyapatite (HA) has been studied recently as a drug carrier in prevent implant infections due to its biocompatibility and osteoconductive properties, but most of these studies failed to control infection. The aim of the present study was to evaluate the potential of this impurity-free novel HA nanotube as a carrier for drug delivery in a controlled manner. Gentamicin was selected as an antibiotic to study the drug-carrier properties of this novel HA nanotube. Gentamicin was introduced into the HA nanotubes through immersion and evaporation process. Gentamicin-loaded HA nanotubes were then placed in phosphate buffered saline (PBS) and drug release profile was then monitored by measuring free genntamicin in the solution. An initial burst release of the drug occurred in the first 24 hours; subsequently, 84.7% of the drug was released from the nanotubes in 9 days. After 13 d, the concentrations of released drug were measured close to 2 µg/ml. The porosity of the gentamicin-loaded HA nanotubes was also observed using a Hitachi s-4800 high-resolution SEM, further confirming the drug-carrier property of HA nanotubes. Our novel bone substitute is an effective prophylactic tool for the local delivery of gentamicin to prevent periprosthetic infections.

Coexistence of multiple rare spinal abnormalities in type 1 neurofibromatosis: a case report and literature review.

Orthopaedic involvement is the most common clinical presentation of Neurofibromatosis type 1 (NF-1) patients with the spinal abnormalities more frequently affected. In the spinal deformities of NF-1 patients, despite the scoliosis is the most frequent finding, several distinctive radiographic features, such as dural ectasia, defective pedicles, and spondylolisthesis, are relatively less common. Here, we reported a 16-year-old boy diagnosed with NF-1 who presented with dural ectasia, defective pedicles, and spondylolisthesis concomitantly, described the surgical treatment and provided a literature review. The boy complained of low back and leg pain for two months. On clinical examination, the patient showed multiple café au lait spots on his back and no neurological deficit. He had a family history of neurofibromatosis as his father suffering from NF-1. Imaging results demonstrated mild scoliosis, posterior scalloping of the lumber spine, L5 spondylolisthesis on plain radiographs, and marked dural ectasia of L3-L5 on MRI. Furthermore, the CT scan showed presence of thin pedicles at L3, bilateral symmetrical pedicle clefts at L4, and pars interarticularis fractures at L5. The patient received a long level posterior fusion from L1 to S1 with pedicle screws. Iliac crest autogenous graft mixed with artificial bone were used to achieve solid arthrodesis. At nine-month follow-up, the patient was asymptomatic and able to live a normal life. Our observation demonstrated that familiarity with those distinctive features in NF-1 patients could be contributed to making an early diagnosis and optimizing treatment.

Efficient induction of specific cytotoxic T lymphocytes against gastric adenocarcinoma by a survivin peptide.

Survivin has been demonstrated to be an excellent target for immunotherapy in several types of cancer, but little is known of the efficacy of survivin with gastric adenocarcinoma. In this study, a simple method was performed, and relatively high efficacy was shown upon inducing survivin-derived peptide-specific cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells of healthy donors. The induced CTLs exhibited specific lysisagainstHLA-A2 matched tumor cells in vitro, and similar results were demonstrated in primary cell cultures isolated from patients with gastric adenocarcinoma. Up to 30% of randomly selected patients could potentially benefit from immunotherapy targeting survivin. These results suggested that this survivin epitope peptide could be a promising vaccine candidate for immunotherapy for patients with gastric adenocarcinoma.

Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma.

AIM: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis. METHODS: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro. RESULTS: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro (47.17% ± 15.61% vs 17% ± 3.6%, P = 0.031) and tumor growth in nude mice (917.86 ± 249.35 mm(3)vs 337.23 ± 124.43 mm(3), P < 0.05). Using flow cytometry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells (P = 0.025). The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts. CONCLUSION: Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma.

AIM: To identify the novel methylation-silenced gene pentraxin 3 (PTX3) in esophageal squamous cell carcinoma (ESCC). METHODS: PTX3 mRNA expression was examined in six human ESCC cell lines, one human immortalized normal esophageal epithelial cell line, primary ESCC tumor tissue, and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction (RT-PCR). Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels. Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene. RESULTS: In the majority of ESCC cell lines, we found that PTX3 expression was down-regulated due to gene promoter hypermethylation, which was further confirmed by bisulphite genomic sequencing. Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines. Methylation was more common in tumor tissues (85%) than in adjacent nontumor tissues (25%) (P < 0 .01). CONCLUSION: PTX3 is down-regulated through promoter hypermethylation in ESCC, and could potentially serve as a biomarker of ESCC.

Identification of GPX3 epigenetically silenced by CpG methylation in human esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common causes of cancer mortality in the gastrointestinal tract. Promoter hypermethylation of tumor suppressor genes contributes to gene inactivation during development of ESCC. AIM: To identify novel methylation-silenced genes in ESCC. METHODS: Genome-wide microarrays were applied to search for genes that were markedly upregulated after treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) and that were markedly decreased in tumor tissue compared with paired adjacent nontumor tissue. Reverse-transcription polymerase chain reaction (PCR), immunohistochemistry, methylation-specific PCR, and bisulfite genomic sequencing were employed to investigate expression and methylation of candidate genes in five human ESCC cell lines, two human immortalized normal esophageal epithelial cell lines, primary ESCC tumor tissues, and paired adjacent nontumor tissues. RESULTS: GPX3 was selected as a novel candidate hypermethylated gene in ESCC through microarray analysis. In most ESCC cell lines, GPX3 messenger RNA (mRNA) expression was downregulated and the CpG island of GPX3 promoter was methylated. Demethylation treatment with 5-Aza-dC restored GPX3 mRNA expression. Methylation of GPX3 promoter was more frequent in ESCC tumor tissues (71.4%) than in adjacent nontumor tissues (10.7%) (P < 0.001), and methylation of GPX3 promoter correlated significantly with GPX3 mRNA downregulation. Finally, GPX3 protein expression was also significantly lower in ESCC tumor tissues than in adjacent nontumor tissues. CONCLUSION: GPX3 is downregulated through promoter hypermethylation in ESCC, which may be a potential biomarker of ESCC.

Peripheral blood monocytes from the decompensated liver cirrhosis could migrate into nude mouse liver with human hepatocyte-markers expression.

Adult stem cells provide a promising alternative for the treatment of decompensated liver cirrhosis. Our previous study showed that peripheral blood monocytes (PBMCs) from decompensated liver cirrhosis could differentiate into hepatocyte-like cells in vitro. We here aimed to investigate the differential potential of the PBMCs under liver environment. PBMCs were isolated from one cirrhotic patient, who was mobilized by the recombinant human granulocyte colony stimulating factor for consecutive 3days, and then PBMCs were transplanted into nude mice by tail vein after labeled with PKH26-GL. The location of the transplanted PBMCs was identified by PKH26-GL staining and PRINS for human SRY gene. The expressions of human hepatocyte-markers were detected by immunohistochemistry, RT-PCR, and Western blot analysis. Our results demonstrated that PBMCs from decompensated liver cirrhosis could migrate into the liver of nude mice with human hepatocyte-markers expression, indicating that autologous PBMCs transplantation might be one alternative therapy for the decompensated liver cirrhosis.

Cell tracing techniques in stem cell transplantation.

Pluripotent stem cells have shown great therapeutic promise because of their natural capacity to regenerate damaged tissue. Likewise, autologous stem cells or genetically modified stem cells have already been successfully applied in animal or clinical experimental studies including cardiopathy, diabetic disease, system lupus erythema, pancreatic disease, and liver disease. In these studies regarding stem cell transplants in different diseases, identifying the location of implanted cells and distinguishing them from endogenous cells is the first and most important step. Moreover, different tracing techniques were applied in different studies for their different sensitivity, dynamic range, convenience and reliability of their assays. Therefore, we will here review different tracing techniques and their applications in stem cell transplants, including both experiment studies and preclinical trials.