Characterizing the relationship between MRI radiomics and AHR expression and deriving a predictive model for prognostic assessment in glioblastoma.

PURPOSE: Aryl hydrocarbon receptor (AHR), a crucial molecular marker associated with glioma, is a potential therapeutic target. We aimed to establish a non-invasive predictive model for AHR through radiomics. METHODS: Contrast-enhanced T1-weighted (T1W) MRI and the corresponding and clinical variables of glioblastoma patients from The Cancer Genome Atlas (TCGA) and The Cancer Imaging Archive (TCIA) were obtained for analysis. KM curves and Cox regression analyses were used to assess the prognostic value of AHR expression. The radiomics features were screened by Max-Relevance and Min-Redundancy (mRMR) and recursive feature elimination (RFE), followed by the construction of two predictive models using logistic regression (LR) and a support vector machine (SVM). RESULTS: The expression levels of AHR in tumour patients were significantly higher than those in the control group, and higher AHR expression was associated with worse prognosis (P<0.05). AHR remained a risk factor for poor prognosis in glioblastoma after multivariate adjustment (HR: 1.61, 95% CI: 1.085-2.39, P<0.05). The radiomics models constructed using LR and SVM based on three selected features achieved area under the curve (AUC) values of 0.887 and 0.872, respectively. Radiomics score emerged as a key factor influencing overall survival (OS) after multivariate adjustment in the Cox model (HR: 3.931, 95% CI: 1.272-12.148, P < 0.05). CONCLUSION: The radiomics models could effectively distinguish the expression levels of AHR and predict prognosis in patients with glioblastoma, which may serve as a powerful tool to assist clinical assessment and precision treatment.

High expression of serine protease inhibitor kazal type 1 predicts poor prognosis and promotes the progression and invasion of oral tongue squamous cell carcinoma.

OBJECTIVE: This study aimed to investigate the expression of serine protease inhibitor kazal type 1 (SPINK1) and its carcinogenic effect in oral tongue squamous cell carcinoma (OTSCC). DESIGN: Initially, bioinformatics analysis was conducted using data from The Cancer Genome Atlas and Gene Expression Omnibus to compare SPINK1 mRNA expression between malignant and adjacent tissues. Subsequently, the impact of differential expression on survival and other clinical variables was examined. Additionally, histology microarray analysis was performed to assess SPINK1 protein expression in 35 cases of malignant and adjacent tissues. Finally, alterations in SPINK1 expression were evaluated to determine its biological phenotypes in OTSCC, including proliferation, apoptosis, invasion, and metastasis. RESULTS: OTSCC tissues exhibit higher levels of SPINK1 compared to surrounding cancerous tissues. Notably, increased SPINK1 expression correlates with the pathological N stage and independently predicts overall survival among patients with OTSCC. CONCLUSION: Suppression of SPINK1 inhibited OTSCC cell proliferation, invasion, and motility while promoting apoptosis. These findings suggest that SPINK1 may serve as a prognostic biomarker as well as a potential therapeutic target for managing OTSCC.

CKS1B as a potential target for prognostic assessment and intervention in pancreatic cancer and its role in abnormal proliferation and cellular phenotype through mediation of cell cycle signaling pathways.

OBJECTIVES: To investigate the role of cell cycle protein-dependent kinase regulatory subunit 1B (CKS1B) in driving the aggressive and rapid proliferation observed in pancreatic cancer. METHODS: A comprehensive analysis was carried out using raw mRNA information and data from 2 databases: the cancer genome atlas and gene expression omnibus. The differential expression of CKS1B at the mRNA and tissue levels in cancer and adjacent paracancerous tissues were assessed. Additionally, the relationship of CKS1B expression and overall survival (OS) rate was investigated using Kaplan-Meier survival curves. Potential molecular mechanisms by which CKS1B may influence the biological characteristics of pancreatic cancer were explored using resources available within the encyclopedia of RNA interactomes database. RESULTS: The CKS1B exhibited significant differential expression at the mRNA as well as protein levels. A correlation with statistical significance between CKS1B expression and N stage, age, and alcohol consumption was observed. Notably, high CKS1B expression was determined as a predictive factor for worse OS. Furthermore, the analysis revealed a potential synergistic role between CKS1B and the molecule PKMYT1, which could impact the ATR-Chk1-Cdc25 signaling pathway and disrupt the G2/M checkpoint within the cell cycle, ultimately promoting abnormal tumor proliferation. CONCLUSION: The CKS1B may serve as a novel potential prognostic factor in pancreatic cancer and is involved in the abnormal proliferation biology phenotype by mediating cell cycle signaling pathways.

Comparison of involved-field intensity-modulated radiotherapy combined with S-1 vs radiotherapy alone for elderly patients with esophageal cancer.

BACKGROUND: It is estimated that about 30% of esophageal cancer (EC) patients are over 70 years old. Therefore, there is less evidence on the diagnosis and management of elderly EC patients. It is important to explore how elderly EC patients benefit from radical radiochemotherapy regimens, including the target area of radiotherapy (RT), radiation dose and fraction, and choice of chemotherapy drugs. AIM: To compare the efficacy of involved-field intensity-modulated RT (IF-IMRT) combined with S-1 vs RT alone in the treatment of elderly EC patients in terms of safety, short-term response, and survival. METHODS: Thirty-four EC patients aged > 70 years were prospectively enrolled between December 2017 and December 2019. Based on the random number table, they were divided into an IF-IMRT + S-1 group and an IF-IMRT alone group, with 17 patients in each group. All patients were treated with IF-IMRT at a dose of 50.4-56 Gy in 28-30 fractions (1.8-2 Gy/fraction, 5 fractions/wk). Oral S-1 was administered concomitantly in the IF-IMRT + S-1 group for 14 consecutive days, and a second cycle was started 7 d after drug withdrawal. After RT, 4 cycles of S-1 treatment were offered as the consolidation chemotherapy. The safety, short-term response, and survival were observed after the treatment. RESULTS: As of April 2022, these 34 patients had been followed up for 15.2-32.5 mo, with a median follow-up period of 24.5 mo. Complete efficacy indicators were obtained from all the patients. The objective response rate was 88.2% vs 76.5%, respectively, in the IF-IMRT + S-1 group and the RT alone group, where as the disease control rate was 100% vs 82.4%, respectively. The incidence of adverse events including grade 1-2 fatigue, granulocytopenia, thrombocytopenia, anemia, radiation esophagitis, radiation-induced skin injury, and radiation-induced lung injury was not significantly different between these two groups, so was the incidence of the grade 3 radiation esophagitis (0% vs 5.7%). The rate of progressive disease (PD) was 52.9% (n = 9) in the IF-IMRT + S-1 group and 64.7% (n = 11) in the RT alone group. The median progression-free survival (PFS) was 23.4 mo vs 16.3 mo, and the 2-year PFS rate was 42% vs 41.2%. The median overall survival (OS) was 27.0 mo vs 23.0 mo, and the 2-year OS rate was 58.8% vs 47.1%. Multivariate analysis showed that age was a significant prognostic factor (P = 0.0019); patients aged < 75 years had a significant survival advantage over patients aged ≥ 75 years. The locations of EC also affected the prognosis. In the IF-IMRT + S-1 group, the number of chemotherapy cycles was a significant prognostic factor (P = 0.0125), and the risk of PD was significantly lower in EC patients who had received 6 cycles of chemotherapy than those who had received 2-5 cycles of chemotherapy. CONCLUSION: Compared with IF-IMRT alone, IF-IMRT + S-1 shows the benefits of preventing PD and prolonging survival without increasing adverse reactions. Therefore, this concurrent radiochemotherapy deserves clinical application.

Implantable Bioresponsive Hydrogel Prevents Local Recurrence of Breast Cancer by Enhancing Radiosensitivity.

Breast cancer is one of the most common types of cancer. Patients are often concerned about regional recurrence after breast cancer surgery. Radiotherapy plays a vital role in reducing recurrence and prolonging the survival of patients undergoing breast-conserving surgery and high-risk mastectomy. However, 8-15% of patients still have disease progression due to radiation resistance. Therefore, new strategies for combination radiotherapy sensitization must be investigated. In this study, an implantable drug loading system, sunitinib nanoparticles @ matrix metalloproteinases -response hydrogel (NSMRH), uses enzyme-sensitive hydrogel as a carrier to load sunitinib nanoparticles, was identified. The releasing profile demonstrated that sunitinib nanoparticles may be continuously released from the hydrogels. Functional experiments revealed that, when paired with NSMRH, radiation may significantly inhibit tumor cell proliferation, migration, and invasion in vitro. Further animal experiments showed that NSMRH combined with radiotherapy could more effectively control the recurrence of subcutaneous xenograft tumors, prolong the survival time, and have no obvious toxicity in nude mice. Finally, by studying the molecular mechanism of NSMRH, it was hypothesized that in breast cancer cells, NSMRH cooperated with sensitized radiotherapy, mainly due to significantly blocking the G2/M phase, reducing the DNA repair efficiency, inhibiting tumor angiogenesis, promoting apoptosis, and reversing the abnormal expression of platelet-derived growth factor receptor alpha (PDGFRA) after radiotherapy. These findings suggest that NSMRH's radiation sensitization and anti-tumor activity may aid in the development of a novel method in future clinical applications.

Correlation of carbonic anhydrase 9 (CA9) with pathological T-stage and prognosis in patients with oral tongue squamous cell carcinoma.

BACKGROUND: We explored the mechanisms underlying tumorigenesis in oral tongue squamous cell carcinoma (OTSCC) with the goal of uncovering prognostic molecular biomarkers. METHODS: An mRNA sequencing dataset was obtained from The Cancer Genome Atlas (TCGA) database, and differentially expressed genes (DEGs) were selected using R language software packages. Functional enrichment analysis was conducted with DAVID software and protein-protein interaction (PPI) networks were constructed using the STRING database. The relationship between hub genes and overall survival (OS) was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression models. Expression of the candidate gene, carbonic anhydrase 9 (CA9), was verified by real-time RT-PCR, western blotting, and immunohistochemistry. RESULTS: DEGs (n=581) were obtained from 11 OTSCC samples and corresponding adjacent non-tumor tissues. Gene ontology (GO) analysis revealed that most DEGs were implicated in anterior/posterior pattern specification, embryonic skeletal system morphogenesis, and multicellular organism development, and pathway analysis suggested that DEGs were associated with neuroactive ligand-receptor interaction, calcium signaling pathway and transcriptional misregulation in the cancer. A PPI network consisting of 301 nodes and 2011 edges was constructed and 71 hub genes, with high degrees of connectivity in the network, were identified. Kaplan-Meier analysis of the hub genes indicated that high expression of CA9, LHX1, and KISS1R and low expression of CCKAR were associated with poor OS in OTSCC; however, only CA9 was a significant prognostic factor influencing survival in OTSCC on multivariate analysis. High expression of CA9 was associated with poor pathological T-stage. CA9 tumor specificity was confirmed using the Gene Expression Omnibus (GEO) database and further molecular tests. CONCLUSIONS: We identified key DEGs that may assist in the molecular understanding of OTSCC. CA9 warrants further exploration as potential prognostic biomarker and therapeutic target in OTSCC.

Integration of Human Umbilical Cord Mesenchymal Stem Cells-Derived Exosomes with Hydroxyapatite-Embedded Hyaluronic Acid-Alginate Hydrogel for Bone Regeneration.

The treatment of bone defects has plagued clinicians. Exosomes, the naturally secreted nanovesicles by cells, exhibit great potential in bone defect regeneration to realize cell-free therapy. In this work, we successfully revealed that human umbilical cord mesenchymal stem cells-derived exosomes could effectively promote the proliferation, migration, and osteogenic differentiation of a murine calvariae preosteoblast cell line in vitro. Considering the long period of bone regeneration, to effectively exert the reparative effect of exosomes, we synthesized an injectable hydroxyapatite (HAP)-embedded in situ cross-linked hyaluronic acid-alginate (HA-ALG) hydrogel system to durably retain exosomes at the defect sites. Then, we combined the exosomes with the HAP-embedded in situ cross-linked HA-ALG hydrogel system to repair bone defects in rats in vivo. The results showed that the combination of exosomes and composite hydrogel could significantly enhance bone regeneration. Our experiment provides a new strategy for exosome-based therapy, which shows great potential in future tissue and organ repair.

The synthesis and application of nano doxorubicin- indocyanine green matrix metalloproteinase-responsive hydrogel in chemophototherapy for head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies, with high rates of mortality and morbidity worldwide. Owing to the special anatomical location of this tumor, an effective, minimally invasive treatment with low systemic toxicity is highly desirable. Hydrogels have shown great potential for tumor-targeting therapy, with excellent performance. However, there have been few reports on co-loading photosensitizers and chemotherapeutic drugs into hydrogels. In this study, we synthesized a nano doxorubicin-indocyanine green matrix metalloproteinase (MMP)-responsive hydrogel (denoted as NDIMH), combining chemotherapy and phototherapy, to achieve superior antitumor efficacy. METHODS: First, NDIMH was synthesized and characterized by scanning electron microscopy and drug-release assays. Second, the photosensitivity properties and antitumor efficiency of this drug delivery system were studied in vivo and in vitro. Last, the imaging and biodistribution of NDIMH were monitored using the Maestro EX in vivo imaging system. RESULTS: The nanodrugs loaded into the smart hydrogel exhibited uniform size distribution, excellent size stability, and a sustained release in the presence of MMP-2. NDIMH showed ideal photosensitivity characteristics under light. NDIMH with 808 nm near-infrared (NIR) irradiation effectively inhibited the viability, invasion, and metastasis of SCC-15 in vitro. After intratumoral injection of NDIMH with 808 nm NIR illumination, the hydrogels exhibited favorable synergistic antitumor efficacy and acceptable biosafety. Additionally, fluorescence imaging showed that NDIMH could significantly improve the retention of nanodrugs at the tumor site. CONCLUSION: The intratumoral injection of NDIMH with 808 nm NIR irradiation could be a promising chemophototherapy alternative for HNSCC.

Triptolide reduces proliferation and enhances apoptosis in drug-resistant human oral cancer cells.

Triptolide (TPL) is a traditional Chinese medicine that possesses anti-multidrug resistance (MDR) properties against various cancers, including oral cancer. However, the functional roles of TPL in oral cancer cells and its potential ability to overcome MDR have not fully evaluated. Therefore, in this study we used oral cancer cell line SAS to establish Taxol-resistant cell line SAS/Taxol and investigated the effects of TPL on MDR, proliferation, and apoptosis of SAS/Taxol cells. We first demonstrated that TPL overcame MDR in SAS/Taxol cells. In addition, TPL induced prominent proliferation inhibition, cell cycle arrest, and apoptosis of SAS/Taxol cells. Furthermore, the pro-apoptotic effect of TPL on SAS/Taxol cells was dependent on intrinsic and extrinsic apoptotic pathways involved in the activation of caspases. Consistently, TPL successfully hampered oral tumor growth by inducing cell apoptosis in a xenograft mouse model. Overall, these results indicated that TPL circumvented MDR of SAS/Taxol cells by inhibition of proliferation and induction of apoptosis which was partly mediated by the intrinsic and extrinsic apoptotic pathways, suggesting the potential therapeutic value of TPL on Taxol-resistant human oral cancer.

MLKL-PITPα signaling-mediated necroptosis contributes to cisplatin-triggered cell death in lung cancer A549 cells.

Necroptosis has been reported to be involved in cisplatin-induced cell death, but the mechanisms underlying the occurrence of necroptosis are not fully elucidated. In this study, we show that apart from apoptosis, cisplatin induces necroptosis in A549 cells. The alleviation of cell death by two necroptosis inhibitors-necrostatin-1 (Nec-1) and necrosulfonamide (NSA), and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) at serine 358, suggest the involvement of receptor-interacting protein kinase 1 (RIPK1)-RIPK3-MLKL signaling in cisplatin-treated A549 cells. Additionally, the initiation of cisplatin-induced necroptosis relies on autocrine tumor necrosis factor alpha (TNF-α). Furthermore, we present the first evidence that phosphatidylinositol transfer protein alpha (PITPα) is involved in MLKL-mediated necroptosis by interacting with the N terminal MLKL on its sixth helix and the preceding loop, which facilitates MLKL oligomerization and plasma membrane translocation in necroptosis. Silencing of PITPα expression interferes with MLKL function and reduces cell death. Our data elucidate that cisplatin-treated lung cancer cells undergo a new type of programmed cell death called necroptosis and shed new light on how MLKL translocates to the plasma membrane.

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Gemcitabine enhances cell invasion via activating HAb18G/CD147-EGFR-pSTAT3 signaling.

Pancreatic cancer, one of the most lethal cancers, has very poor 5-year survival partly due to gemcitabine resistance. Recently, it was reported that chemotherapeutic agents may act as stressors to induce adaptive responses and to promote chemoresistance in cancer cells. During long-term drug treatment, the minority of cancer cells survive and acquire an epithelial-mesenchymal transition phenotype with increased chemo-resistance and metastasis. However, the short-term response of most cancer cells remains unclear. This study aimed to investigate the short-term response of pancreatic cancer cells to gemcitabine stress and to explore the corresponding mechanism. Our results showed that gemcitabine treatment for 24 hours enhanced pancreatic cancer cell invasion. In gemcitabine-treated cells, HAb18G/CD147 was up-regulated; and HAb18G/CD147 down-regulation or inhibition attenuated gemcitabine-enhanced invasion. Mechanistically, HAb18G/CD147 promoted gemcitabine-enhanced invasion by activating the EGFR (epidermal growth factor receptor)-STAT3 (signal transducer and activator of transcription 3) signaling pathway. Inhibition of EGFR-STAT3 signaling counteracted gemcitabine-enhanced invasion, and which relied on HAb18G/CD147 levels. In pancreatic cancer tissues, EGFR was highly expressed and positively correlated with HAb18G/CD147. These data indicate that pancreatic cancer cells enhance cell invasion via activating HAb18G/CD147-EGFR-pSTAT3 signaling. Our findings suggest that inhibiting HAb18G/CD147 is a potential strategy for overcoming drug stress-associated resistance in pancreatic cancer.

A novel small-molecule compound targeting CD147 inhibits the motility and invasion of hepatocellular carcinoma cells.

CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression.

CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-ß-catenin-WAVE2 signaling.

The acquisition of inappropriate migratory feature is crucial for tumor metastasis. It has been suggested that CD147 and Annexin A2 are involved in regulating tumor cell movement, while the regulatory mechanisms are far from clear. In this study, we demonstrated that CD147 physically interacted with the N-terminal domain of Annexin A2 and decreased Annexin A2 phosphorylation on tyrosine 23. In vitro kinase assay showed that the I domain of CD147 was indispensable for CD147-mediated downregulation of Annexin A2 phosphorylation by Src. Furthermore, we determined that p-Annexin A2 promoted the expression of dedicator of cytokinesis 3 (DOCK3) and DOCK3 blocked ß-catenin nuclear translocation, resulting in inhibition of ß-catenin signaling. In addition, DOCK3 inhibited lamellipodium dynamics and tumor cell movement. Also, we found that ß-catenin signaling increased WAVE2 expression. Therefore, DOCK3 was characterized as a negative regulator of WAVE2 expression via inhibiting ß-catenin signaling. Our study provides the first evidence that CD147 promotes tumor cell movement and metastasis via direct interaction with Annexin A2 and DOCK3-ß-catenin-WAVE2 signaling axis.

Necrostatin-1 reduces intestinal inflammation and colitis-associated tumorigenesis in mice.

Necroptosis, a novel form of programmed cell death, was recently shown to be strongly associated with intestinal inflammation in mice and in pediatric patients with inflammatory bowel disease (IBD). Persistent inflammation of the colon is an important risk factor for colorectal cancer. Necrostatin-1 (Nec-1), known as a specific inhibitor of necroptosis, through preventing the receptor-interacting protein (RIP) 1 and RIP3 interaction. In the present study, the anti-inflammatory and antitumorigenic efficacy of necrostatin-1 was studied in mouse models of colitis and colitis-associated cancer (CAC). We found that in acute dextran sulfate sodium (DSS)-induced colitis, treatment with necrostatin-1 significantly suppressed colitis symptoms in mice, including weight loss, colon shortening, colonic mucosa damage and severity, and excessive production of interleukin-6. Necrostatin-1 administration inhibited the upregulation of RIP1 and RIP3 and enhanced the expression of caspase-8 in DSS-induced colitis. In addition, the anti-inflammatory effect of necrostatin-1 was confirmed by in vitro analyses. Necrostatin-1 treatment reduced the production of proinflammatory cytokine and extracellular HMGB1 release in HT-29 cells in active necroptosis. Furthermore, In a mouse model of colitis-associated tumorigenesis, necrostatin-1 administration significantly suppressed tumor growth and development through inhibiting JNK/c-Jun signaling. Taken together, these findings suggest that necrostatin-1 might be a promising therapeutic option for the treatment of colitis-associated colorectal cancer in patients with IBD.

Cytoplasmic EpCAM over-expression is associated with favorable clinical outcomes in pancreatic cancer patients with Hepatitis B virus negative infection.

The identification of reliable prognostic markers that distinguish patients' status and predict therapeutic response can improve the clinical outcomes of pancreatic cancer patients. The epithelial cell adhesion molecule (EpCAM) is known to be highly expressed in cancers and serves as a prognosis factor. Generally, membranous EpCAM expression in cancer cells and its clinical significance are evaluated. However, there is also an evidence of cytoplasmic EpCAM distribution in cancer cells. Hence, we investigated which kind of the immunostaining pattern in pancreatic cancer patients was, and whether membranous or cytoplasmic immunostaining had clinical significance. We determined the cytoplasmic or membranous EpCAM expression by a well-established immunohistochemical staining protocol in 157 pairs of carcinoma and paired adjacent non-tumor pancreatic tissue samples using the EpCAM-specific antibody. Furthermore, we evaluated the relationship between tumoral EpCAM expression of resected specimens and patient's overall survival as well as other biological variables like clinical prognosis by Kaplan-Meier method and χ(2) test. We found that pancreatic cancer patients had expressed higher level of cytoplasmic EpCAM but lower level of membranous EpCAM, and their expressions were significantly correlated. Cytoplasmic EpCAM acted as a favorable prognosis factor on survival time in patients with HBV negative infection. Pancreatic cancer patients with cytoplasmic EpCAM over-expression and negative Hepatitis B virus infection might benefit further from post-surgery chemotherapy. These data suggested a potential role of cytoplasmic EpCAM in predicting patient's prognosis and determining therapeutic strategy.

CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cells.

Metastasis is considered a dynamic process in tumor development that is related to abnormal migration and invasion. Tumor cells can move as individual cells in two interconvertible modes: mesenchymal-type and amoeboid. Previously, we reported that the interaction between CD147 and Annexin II can inhibit the amoeboid movement in hepatocellular carcinoma (HCC) cells. However, the mechanism of CD147 involved in mesenchymal movement is still unclear. Notably, our results show overexpression of CD147 led to mesenchymal-type movement in HCC cells. Evidence indicated that the mesenchymal-type cell movement induced by CD147 was Src dependent, as observed by confocal microscopy and Rac1 activity assay. The phosphorylation of Src (pY416-Src) can be up-regulated by CD147, and this regulation is mediated by focal adhesion kinase (FAK). Next, we identified DOCK8 as a GEF for Rac1, a key molecule driving mesenchymal-type movement. We also found that Src promotes STAT3 phosphorylation and STAT3 facilitates DOCK8 transcription, thus enhancing DOCK8 expression and Rac1 activation. This study provides a novel mechanism of CD147 regulating mesenchymal-type movement in HCC cells.

Enhancement of CD147 on M1 macrophages induces differentiation of Th17 cells in the lung interstitial fibrosis.

Lung interstitial fibrosis is a chronic lung disease, and few effective therapies are available to halt or reverse the progression of the disease. In murine and human lung fibrosis, the expression of CD147 is increased. However, the role of CD147 in lung fibrosis has not been identified, and it remains to be determined whether lung fibrosis would be improved by decreasing the expression of CD147. A murine bleomycin-induced lung interstitial fibrosis model was used in the experiments, and HAb18 mAbs and CsA were administered during the induction of lung fibrosis. In our study, we found that the HAb18 mAbs markedly reduced the collagen score and down-regulated M1 macrophages and Th17 cells. In vitro, flow cytometry analysis showed that M1 macrophages induced higher Th17 differentiation than M2 macrophages. After treatment with HAb18 mAbs or after reducing the expression of CD147 by lentivirus interference in M1 macrophages, the level of Th17 cells were significantly inhibited. In conclusion, HAb18 mAbs or CsA treatment ameliorates lung interstitial fibrosis. CD147 promoted M1 macrophage and induced the differentiation of Th17 cells in lung interstitial fibrosis, perhaps by regulating some cytokines such as IL-6, IL-1ß, IL-12 and IL-23. These results indicated that CD147 may play an important role in the development of lung interstitial fibrosis.

Inhibition of synovitis and joint destruction by a new single domain antibody specific for cyclophilin A in two different mouse models of rheumatoid arthritis.

INTRODUCTION: Cyclophilin A (CypA) is implicated in rheumatoid arthritis (RA) pathogenesis. We studied whether a novel anti-CypA single domain antibody (sdAb) treatment would modulate the severity of the disease in two different animal models of RA. METHODS: A novel sdAb, named sdAbA1, was screened from an immunized camel sdAb library and found to have a high binding affinity (KD = 6.9 × 10-9 M) for CypA. The SCID-HuRAg model and the collagen-induced arthritis (CIA) in mice were used to evaluate the effects of sdAbA1 treatment on inflammation and joint destruction. For in vitro analysis, monocytes/macrophages were purified from synovial fluid and peripheral blood of patients with RA and were tested for the effect of anti-CypA sdAb on metalloproteinase (MMP) production. Human monocyte cell line THP-1 cells were selected and western blot analyses were performed to examine the potential signaling pathways. RESULTS: In the CIA model of RA, the sdAbA1 treatment resulted in a significant decrease in clinical symptoms as well as of joint damage (P <0.05). In the SCID-HuRAg model, treatment with anti-CypA antibody sdAbA1 significantly reduced cartilage erosion, inflammatory cell numbers and MMP-9 production in the implanted tissues (P <0.05). It also significantly reduced the levels of human inflammatory cytokines IL-6 and IL-8 in mouse serum (P <0.05). No toxic effects were observed in the two animal models. In vitro results showed that sdAbA1 could counteract CypA-dependent MMP-9 secretion and IL-8 production by interfering with the ERK-NF-κB pathway. CONCLUSIONS: Blockade of CypA significantly inhibited synovitis and cartilage/bone erosion in the two tested animal models of RA. Our findings provide evidence that sdAbA1 may be a potential therapeutic agent for RA.

The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells.

Endostatin, the C-terminal fragment of collagen XVIII, is a potent anti-angiogenic factor that significantly modulates the gene expression pattern in endothelial cells. Upon cell surface binding, endostatin can not only function extracellularly, but also translocate to the nucleus within minutes. However, the mechanism by which this occurs is partially understood. Here we systematically investigated the nuclear translocation mechanism of endostatin. By chemical inhibition and RNA interference, we firstly observed that clathrin-mediated endocytosis, but not caveolae-dependent endocytosis or macropinocytosis, is essential for the nuclear translocation of endostatin. We then indentified that nucleolin and integrin α5ß1, two widely accepted endostatin receptors, mediate this clathrin-dependent uptake process, which also involves urokinase plasminogen activator receptor (uPAR). Either mutagenesis study, fluorescence resonance energy transfer assay, or fluorescence cell imaging demonstrates that nucleolin and integrin α5ß1 interact with uPAR simultaneously upon endostatin stimulation. Blockade of uPAR decreases not only the interaction between nucleolin and integrin α5ß1, but also the uptake process, suggesting that the nucleolin/uPAR/integrin α5ß1 complex facilitates the internalization of endostatin. After endocytosis, nucleolin further regulates the nuclear transport of endostatin. RNA interference and mutational analysis revealed that the nuclear translocation of endostatin involves the association of nucleolin with importin α1ß1 via the nuclear localization sequence. Taken together, this study reveals the pathway by which endostatin translocates to the nucleus and the importance of nucleolin in this process, providing a new perspective for the functional investigation of the nuclear-translocated endostatin in endothelial cells.