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Purpose To evaluate treatment outcomes of de novo metastatic nasopharyngeal carcinoma (mNPC) patients receiving taxane/gemcitabine-containing chemotherapy followed by locoregional intensity-modulated radiotherapy (IMRT) and analyze potential prognostic factors. Methods A total of 118 patients between March 2008 and November 2018 were retrospectively analyzed. All the patients were treated with taxane/gemcitabine-containing systemic chemotherapy followed by definitive locoregional IMRT. Potential prognostic factors including baseline absolute lymphocyte count (ALC) and the subdivision of metastasis were analyzed. Results The median follow-up time for the whole group was 31.5 months (range 5–138 months). Of the 118 patients, 9 (7.6%) patients experienced local regional failure and 60 (50.8%) patients had progression of distant metastasis. At the time of the last follow-up, 61 (51.7%) patients were dead. The 5-year actuarial progression free survival (PFS), overall survival (OS),distant metastasis relapse free survival (DMFS) and local regional recurrence free survival (LRFS) were 34.2%, 44%, 41.1% and 82.6%, respectively. Baseline lymphocyte count ≥ 1600/μl prior to the treatment conferred better locoregional control (5y-LRFS 96% vs. 64.7%, p p = 0.023). The multivariate analysis showed that high lymphocyte count was the most relevant predictor of superior PFS (HR = 0.236, p p = 0.04). M subdivision was found as another independent prognostic factor for OS but not for PFS. Conclusion Taxane/gemcitabine-containing chemotherapy combined with IMRT represents an effective treatment modality for mNPC. Baseline ALC is an independent significant prognostic factor for PFS and OS.

The aim of the study was to report long-term results of intensity-modulated radiotherapy for patients with T4 classification nasopharyngeal carcinoma (NPC). From September 2007 to January 2013, 155 patients were retrospectively analyzed. The estimated 10-year local recurrent-free survival (LRFS), regional recurrent-free survival (RRFS), distant metastasis-free survival (DMFS), and overall survival (OS) rates were 79.4%, 93.2%, 69.0%, and 54.2%, respectively. Cycle number of chemotherapy was a significant predictor of LRFS, OS, and progression-free survival. There was no significant difference in survival rates between patients treated with induction chemotherapy (IC) plus concurrent chemoradiotherapy (CCRT) and patients with IC plus IMRT and adjuvant chemotherapy (AC).

To improve the radiation therapy outcomes by inducing more toxicity for tumors and less for normal tissue and switch microenvironment immunosuppressive caused by expression of PD-L1 and tumor-associated macrophages (TAMs) to immunoreactive, we designed a PD-L1-targeted nanoplatform consisting of gold nanoparticles (AuNPs) and superparamagnetic iron oxide nanoparticles (SPIOs)( antiPD-L1-SPIOs@PLGA@Au). AntiPD-L1-SPIOs@PLGA@Au targeted on the B16F10 cell membrane and in cytoplasm, and the association could be reduced by the antibody blocking. In vivo T2-weight image, the best enhancement effect of tumor was achieved two hours after intravenous injection of antiPD-L1-SPIOs@PLGA@Au. Compared with radiation alone, the Do sensitization ratios of antiPD-L1-SPIOs@PLGA@Au was 1.34. The inhibition effect of irradiation combined with antiPD-L1-SPIOs@PLGA@Au was higher than that of radiotherapy alone in the B16F10 subcutaneous tumor model. During the entire observation period, all mice in the antiPD-L1-SPIOs@PLGA@Au group lived longer than 3 weeks. AntiPD-L1-SPIOs@PLGA@Au enhancing radiosensitivity was achieved by increasing ROS production and attenuating DNA damage repair. In vitro and in vivo experiments showed that antiPD-L1-SPIOs@PLGA@Au could promote the polarization of tumor-associated macrophages (TAMs) to M1 and reverse the immunosuppressive regulation induced by TAMs. antiPD-L1-SPIOs@PLGA@Au could increase the expression of CRT in tumor, block the PD-L1/PD pathway, activate CD8+T cells, promote the increase of IFN-γ, TNF-α and IL-12 release, and finally activate the anti-tumor immune response. In conclusion, antiPD-L1-SPIOs@PLGA@Au was proved to have the ability to form targeted imaging and enhance radiosensitivity, block the PD-L1/PD-1 immune checkpoint pathway, reverse the immunosuppressive regulation caused by TAMs, successfully realizing the synergistic effect of radiotherapy combined with immunotherapy.

Background The aim of the study is to identify clinical and dosimetric factors that could predict the risk of hypothyroidism in nasopharyngeal carcinoma (NPC) patients following intensity-modulated radiotherapy (IMRT). Methods A total of 404 non-metastatic NPC patients were included in our study. All patients were treated with IMRT. The thyroid function were performed for all patients before and after radiation at regular intervals. The time onset for developing hypothyroidism was defined as the time interval between the completion of RT and the first recorded abnormal thyroid hormone test. The cumulative incidence rates of hypothyroidism were estimated using Kaplan–Meier method. Univariate and multivariate Cox regression analyses were performed to detect the most promising factors that were associated with hypothyroidism. Results Median follow up was 60.6 months. The 3-, 5- and 7- year cumulative incidence rate of hypothyroidism was 39.4%, 49.1% and 54.7%, respectively. The median time to primary hypothyroidism and central hypothyroidism were 15.4 months (range 2.9–83.8 months) and 29.9 months (range 19.8–93.6 months), respectively. Univariate and multivariate analyses revealed that younger age, female gender and small thyroid volume were the most important factors in predicting the risk of hypothyroidism. Dtmean (mean dose of thyroid), V30-V50 (percentage of thyroid volume receiving a certain dose level) and VS45-VS60 (the absolute volumes of thyroid spared from various dose levels) remained statistically significant in multivariate analyses. Cutoff points of 45 Gy (Dtmean), 80% (Vt40) and 5 cm3 (VS45Gy) were identified to classify patients as high-risk or low-risk group. Conclusion Thyroid Vt40 highly predicted the risk of hypothyroidism after IMRT for NPC patients. We recommended plan optimization objectives to reduce thyroid Vt40 to 80%. Trial registration: Retrospectively registered.

This study aimed to develop prognosis signatures through a radiomics analysis for patients with nasopharyngeal carcinoma (NPC) by their pretreatment diagnosis magnetic resonance imaging (MRI). A total of 208 radiomics features were extracted for each patient from a database of 303 patients. The patients were split into the training and validation cohorts according to their pretreatment diagnosis date. The radiomics feature analysis consisted of cluster analysis and prognosis model analysis for disease free-survival (DFS), overall survival (OS), distant metastasis-free survival (DMFS) and locoregional recurrence-free survival (LRFS). Additionally, two prognosis models using clinical features only and combined radiomics and clinical features were generated to estimate the incremental prognostic value of radiomics features. Patients were clustered by non-negative matrix factorization (NMF) into two groups. It showed high correspondence with patients’ T stage (p

Purpose To evaluate the clinical characteristics and prognosis of elderly nasopharyngeal carcinoma (NPC) patients receiving intensity-modulated radiotherapy (IMRT). Methods From June 2008 to October 2014, 148 newly diagnosed non-metastatic elderly NPC patients (aged ≥ 70 years) receiving IMRT were recruited. Comorbid condition was evaluated using the age-adjusted Charlson Comorbidity Index (ACCI). Kaplan–Meier method was used to estimate survival rates and the differences were compared using log-rank test. Hazard ratio (HR) and the associated 95% confidence interval (CI) were calculated using Cox proportional hazard model by means of multivariate analysis. Results The median follow-up time was 66.35 months. Estimated OS rate at 5 years for the entire group was 61.8% (95% confidence interval [CI] 0.542–0.703). The 5-year OS rate of RT alone group was 58.4% (95% [CI] 0.490–0.696) compared with 65.2% (95% [CI] 0.534–0.796) in CRT group (p = 0.45). In patients receiving IMRT only, ACCI score equal to 3 was correlated with superior 5-year OS rate in comparison with higher ACCI score 62.1% (95% [CI] 0.510–0.766) to 48.5% (95% [CI] 0.341–0.689), respectively; p = 0.024). A 5-year OS rate of 63.1% (95% [CI] 0.537–0.741) was observed in patients younger than 75 years old compared with 57.5% (95% [CI] 0.457–0.723) in patients older (p = 0.026). Patients with early-stage disease (I–II) showed better prognosis than patients with advanced-stage (III–IV) disease (5-year OS, 72.3–55.4%, respectively; p = 0.0073). The Cox proportional hazards model suggested that age independently predicted poorer OS (HR, 1.07; 95%CI 1.00–1.15, p = 0.04). Conclusion The survival outcome of patients aged ≥ 70 years receiving IMRT only was similar to chemoradiotherapy with significantly less acute toxicities. Among the population, age is significantly prognostic for survival outcomes.

Lu Di,1–3,* Kai Qian,2,4,* Chengrun Du,1,2 Chunying Shen,1,2 Ruiping Zhai,1,2 Xiayun He,1,2 Xiaoshen Wang,1,2 Tingting Xu,1,2 Chaosu Hu,1,2 Hongmei Ying1,2 1Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai 200032, China; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China; 3Department of Oncology, Liyang People’s Hospital, Changzhou, Jiangsu 213000, China; 4Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China *These authors contributed equally to this work Background: Salivary duct carcinoma (SDC) is an extremely rare and highly malignant carcinoma, and surgical radical resection is the most effective therapy. However, there were quite a proportion of patients receiving non-radical resections, and how to treat them remained controversial. Thus, the aim of this study is to evaluate whether postoperative radiotherapy could be a salvage treatment of SDC in major salivary glands without radical operations.Patients and methods: We identified 40 pathologically diagnosed SDC patients who came to our hospital and did not receive radical operations. Thirty-three patients received at least one treatment (remedial operation, postoperative radiotherapy and chemotherapy), and seven patients only chose observation and received no further treatment. The prognostic indicators of the local–regional control (LRC) and distant disease-free survival were analyzed using the Kaplan–Meier methods and the Cox proportional hazards regression models.Results: Thirteen patients experienced local–regional recurrence or local progression, and distant metastases were observed in 15 patients. Through multivariate analysis, we found that postoperative radiotherapy was associated with better LRC, but this kind of treatment did not show significant efficacy in the prevention of distant metastasis.Conclusion: SDC is a rare, aggressive malignancy, and a substantial proportion of these patients experienced inadequate initial treatments. Although postoperative radiotherapy could not decrease distant metastases, it might help to improve LRC in patients with SDC. Keywords: salivary duct carcinoma, prognosis, non-radical resection, postoperative radiotherapy

Objective: To analyze the prognostic value of pre-treatment serum lactate dehydrogenase (SLDH) level in patients with nasopharyngeal carcinoma (NPC) receiving intensity-modulated radiotherapy (IMRT) with or without chemotherapy. Methods: From January 2010 to March 2013, 427 eligible patients were reviewed. Pre-treatment SLDH level was measured within 2 weeks prior to treatment. Receiver operating characteristic (ROC) curve analysis was performed to select the optimal cutoff point. The impact of pre-treatment SLDH on overall survival (OS), progression-free survival (PFS) and distant metastasis-free survival (DMFS) were analyzed using Kaplan-Meier method and Cox proportional hazards model. Further propensity score matching was carried out to adjust bias. Results: The optimal cutoff point of 168.5 IU/L was selected based on ROC curve analysis. Multivariate analysis showed that high pre-treatment SLDH level was an independent prognostic factor for OS (P=0.001), PFS (P=0.004) and DMFS (P=0.001). After propensity score matching was performed, it remained to be significantly associated with poor OS (P=0.009), PFS (P=0.015) and DMFS (P=0.008) in the adjusted model. Conclusion: High pre-treatment SLDH level predicts poor survival in patients with NPC treated with IMRT-based therapy. As a routinely performed biomarker, pre-treatment SLDH can be utilized in combination with current Tumor-Node-Metastasis staging to predict survival and to plan a personalized treatment in these patients.

Background: The Cox proportional hazards (CPH) model is the most commonly used statistical method for nasopharyngeal carcinoma (NPC) prognostication. Recently, machine learning (ML) models are increasingly adopted for this purpose. However, only a few studies have compared the performances between CPH and ML models. This study aimed at comparing CPH with two state-of-the-art ML algorithms, namely, conditional survival forest (CSF) and DeepSurv for disease progression prediction in NPC. Methods: From January 2010 to March 2013, 412 eligible NPC patients were reviewed. The entire dataset was split into training cohort and testing cohort in a ratio of 90%:10%. Ten features from patient-related, disease-related, and treatment-related data were used to train the models for progression-free survival (PFS) prediction. The model performance was compared using the concordance index (c-index), Brier score, and log-rank test based on the risk stratification results. Results: DeepSurv (c-index = 0.68, Brier score = 0.13, log-rank test p = 0.02) achieved the best performance compared to CSF (c-index = 0.63, Brier score = 0.14, log-rank test p = 0.38) and CPH (c-index = 0.57, Brier score = 0.15, log-rank test p = 0.81). Conclusions: Both CSF and DeepSurv outperformed CPH in our relatively small dataset. ML-based survival prediction may guide physicians in choosing the most suitable treatment strategy for NPC patients.

// Chengrun Du 1 , Hongmei Ying 1 , Youwang Zhang 1 , Yafang Huang 2 , Ruiping Zhai 1 and Chaosu Hu 1 1 Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, P.R. China 2 Department of Ultrasound, Fudan University Shanghai Cancer Center, Shanghai, P.R. China Correspondence to: Hongmei Ying, email: yinghongmei2011@sina.com Keywords: retropharyngeal nodes, undifferentiated squamous cell carcinoma, transoral sonography-guided fine-needle aspiration, primary unknown, radiotherapy Received: September 18, 2016 Accepted: February 21, 2017 Published: March 18, 2017 ABSTRACT Background and Objective: To evaluate treatment outcomes for patients with retropharyngeal metastatic undifferentiated squamous cell carcinoma (SCC) from an unknown primary site. Methods: From January 2005 to January 2015, patients who presented with enlarged retropharyngeal nodes underwent transoral sonography-guided fine-needle aspiration to confirm histology. Those with metastatic undifferentiated SCC with unknown primary tumors were treated with radical radiotherapy to nasopharyngeal mucosa plus bilateral neck. Chemotherapy was administered for patients staged N2-3. Endpoints included metastatic nodes control, the appearance of primary tumor, overall survival and treatment-related toxicities. Results: A total of 49 patients were recruited into this study. Retropharyngeal and cervical nodal disease was controlled in 96% of all patients. The incidence of occult primary cancer appearance was 8%. No primary cancer other than of the nasopharynx was detected during the course of follow-up. Ten patients developed distant metastases. The 5-year overall survival, progression-free survival, regional relapse free survival, distant metastasis free survival were 79.6%, 61.1%, 83.4%, 73.8%, respectively. Common late adverse effects included xerostomia (57%) and hearing impairment (35%). Conclusion: Radical radiotherapy to both the nasopharynx and bilateral neck can achieve excellent outcome with mild toxicities for patients with retropharyngeal metastatic undifferentiated squamous cell carcinoma from an unknown primary site.

// Fangfang Kong 1,2 , Hongmei Ying 1,2 , Ruiping Zhai 1,2 , Chengrun Du 1,2 , Shuang Huang 1,2,3 , Junjun Zhou 1,2 , Xiayun He 1,2 , Chaosu Hu 1,2 , Zhuoying Wang 2,4 , Tuanqi Sun 2,4 and Qinghai Ji 2,4 1 Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, P.R. China 2 Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, P.R. China 3 Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou, P. R. China 4 Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai, P.R. China Correspondence to: Hongmei Ying, email: // Keywords : intensity-modulated radiotherapy; IMRT; radiotherapy; carcinoma showing thymus-like differentiation; CASTLE Received : March 15, 2016 Accepted : September 02, 2016 Published : September 08, 2016 Abstract Purpose: To evaluate the efficacy and toxicity of adjuvant intensity-modulated radiotherapy (IMRT) after surgery for carcinoma showing thymus-like differentiation (CASTLE). Methods: Between September 2008 and June 2015, 14 CASTLE patients were retrospectively enrolled. The clinical features, treatment procedure and clinical outcomes were reviewed. All patients received postoperative IMRT. The radiation doses ranged from 56Gy/28 fractions to 66Gy/33 fractions. Treatment-related toxicities were graded by National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 3.0. Results:After a median follow-up period of 42 months, only one patient suffered local recurrence and distant metastasis. The most frequently seen acute toxicities were mucositis and dermatitis (grade 1-2). No grade 3-4 toxicities were observed. Conclusions: Although based upon a small series of consecutively treated patients, our study showed that adjuvant IMRT provides satisfactory local-regional control for CASTLE, with acceptable toxicities. Further studies are still warranted to clarify our findings.

Ronald Wihal Oei,1,2,* Lulu Ye,1,2,* Fangfang Kong,1,2 Chengrun Du,1,2 Ruiping Zhai,1,2 Tingting Xu,1,2 Chunying Shen,1,2 Xiaoshen Wang,1,2 Xiayun He,1,2 Lin Kong,1,2 Chaosu Hu,1,2 Hongmei Ying1,2 1Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China; 2Department of Oncology, Shanghai Medical College, Shanghai, People’s Republic of China *These authors contributed equally to this work Purpose: The aim of this article is to investigate the significance of pretreatment prognostic nutritional index (PNI), systemic immune-inflammation index (SII), and their combination in nasopharyngeal carcinoma (NPC) patients receiving intensity-modulated radiotherapy (IMRT).Materials and methods: A total of 585 patients were included. PNI and SII were calculated within 2 weeks prior to treatment. The optimal cutoff points were determined based on receiver operating characteristics curve analysis. The correlation between variables was analyzed. Kaplan–Meier method and Cox proportional hazards model were performed to evaluate the impact of both indices on overall survival (OS), progression-free survival (PFS) and distant metastasis-free survival (DMFS). Further propensity score matching (PSM) was carried out to minimize the effects of confounders. Results: The optimal cutoff point of 53.0 for PNI and 527.20 for SII were selected. Pearson correlation coefficient showed an inverse correlation between PNI and SII (r = –0.232, P < 0.001). Multivariate analysis demonstrated that pretreatment PNI was an independent prognostic factor for OS (P = 0.047) and DMFS (P = 0.002) while pretreatment SII was an independent prognostic factor for OS (P = 0.003), PFS (P = 0.002), and DMFS (P = 0.002). After PSM, both parameters remained as independent prognosticators of survival. Additional prognostic value was observed in the combined use of PNI and SII.Conclusion: Pretreatment PNI and SII are promising indicators of survival in NPC patients undergoing IMRT. They can be utilized to refine current TNM staging system in predicting prognosis and developing an individualized treatment in these patients. Keywords: prognostic nutritional index, systemic immune-inflammation index, nasopharyngeal carcinoma, intensity-modulated radiotherapy, prognostic factor, survival

Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas. In this study, wound-healing, transwell migration and matrigel invasion assays showed that MUC1 promotes human hepatocellular carcinoma (HCC) cell migration and invasion by MUC1 gene silencing and overexpressing. Treatment with exogenous transforming growth factor beta (TGF-β)1, TGF-β type I receptor (TβRI) inhibitor, TGF-β1 siRNAs, or activator protein 1 (AP-1) inhibitor to MUC1-overexpressing HCC cells revealed that MUC1-induced autocrine TGF-β via JNK/AP-1 pathway promotes the cell migration and invasion. In addition, the migration and invasion of HCC cells were more significantly inhibited by JNK inhibitor compared with that by TβRI inhibitor or TGF-β1 siRNAs. Further studies demonstrated that MUC1-mediated JNK activation not only enhances the phosphorylation of Smad2 C-terminal at Ser-465/467 site (Smad2C) through TGF-β/TβRI, but also directly enhances the phosphorylation of Smad2 linker region at Ser-245/250/255 site (Smad2L), and then both of them collaborate to upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of HCC. These results indicate that MUC1 is an attractive target in liver cancer therapy.

// Qiongshu Li 1 , Guomu Liu 1 , Hongyan Yuan 1 , Juan Wang 1 , Yingying Guo 1 , Tanxiu Chen 1 , Ruiping Zhai 1 , Dan Shao 1 , Weihua Ni 1 , Guixiang Tai 1 1 Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130021, China Correspondence to: Guixiang Tai, e-mail: taiguixiang@163.com Keywords: MUC1, pSmad3L, pSmad3C, HCC, JNK Received: November 14, 2014 Accepted: December 16, 2014 Published: January 20, 2015 ABSTRACT Mucin1 (MUC1) is a transmembrane glycoprotein that acts as an oncogene in human hepatic tumorigenesis. Hepatocellular carcinoma (HCC) cells often gain advantage by reducing the tumor-suppressive activity of transforming growth factor beta (TGF-β) together with stimulation of its oncogenic activity as in MUC1 expressing HCC cells; however, molecular mechanisms remain largely unknown. Type I TGF-β receptor (TβRI) and c-Jun NH2-terminal kinase (JNK) differentially phosphorylate Smad3 mediator to create 2 phosphorylated forms: COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Here, we report that MUC1 overexpression in HCC cell lines suppresses TβRI-mediated pSmad3C signaling which involves growth inhibition by up-regulating p21 WAF1 . Instead, MUC1 directly activates JNK to stimulate oncogenic pSmad3L signaling, which fosters cell proliferation by up-regulating c-Myc. Conversely, MUC1 gene silencing in MUC1 expressing HCC cells results in preserved tumor-suppressive function via pSmad3C, while eliminating pSmad3L-mediated oncogenic activity both in vitro and in vivo . In addition, high correlation between MUC1 and pSmad3L/c-Myc but not pSmad3C/p21 WAF1 expression was observed in HCC tissues from patients. Collectively, these results indicate that MUC1 shifts Smad3 signaling from a tumor-suppressive pSmad3C/p21 WAF1 to an oncogenic pSmad3L/c-Myc pathway by directly activating JNK in HCC cells, suggesting that MUC1 is an important target for HCC therapy.

Human mucin 1 (MUC1) is a target for immunotherapy. The major problem associated with MUC1‑based cancer vaccines is the weakness of the immunogenicity of MUC1. The present study aimed to develop an efficient cancer vaccine through generating a recombinant fusion protein consisting of MUC1 and maltose‑binding protein (MBP) by inserting seven tandem repeats encoding the human MUC1 gene into the pMAL‑c2 expression vector. Bacillus Calmette‑Guerin (BCG) was used as an adjuvant. MUC1 was found to predominantly induce T helper type 2 (Th2) cell responses. MUC1/BCG and MUC1‑MBP were found to generate T helper (Th) type 1 and 2 responses, while MUC1‑MBP/BCG induced a Th1 immune profile and stimulated MUC1‑specific cytotoxic T lymphocyte killing activity. MUC1‑MBP, as well as MBP and BCG alone were found to induce natural killer (NK) cell activity, with MUC1‑MBP/BCG observed to synergistically induce NK cell activity. Furthermore, MUC1‑MBP/BCG significantly inhibited MUC1+ B16 cell growth in mice. These findings show that MBP augments the immunogenicity of MUC1 and that BCG enhances the efficacy of the MUC1‑MBP vaccine. Thus, MUC1‑MBP/BCG may have potential as a cancer vaccine for clinical application.

Maltose-binding protein (MBP), a component of the maltose transport system of Escherichia coli, has been commonly thought to have minimal bioactivity. Our previous studies demonstrated that MBP could significantly enhance Bacillus Calmette-Guerin (BCG)-induced T helper 1 (Th1) cell activation in mice. In the present study, we analyzed the effect of MBP on mouse T cells and found that MBP promoted the proliferation and IFN-γ production of CD4(+) T cells, suggesting that MBP directly induces Th1 activation. To explore the mechanism of Th1 activation, the expression of Toll-like receptor 2/4 (TLR2/4) on purified mouse CD4(+) T cells was detected. The results showed that MBP up-regulated TLR2 while down-regulated TLR4 expression, accompanied by a clear increase in MyD88 expression and IκB phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4(+) T cells proliferation, IFN-γ secretion and MyD88 expression, whereas the addition of anti-TLR4 antibody exhibited a contradictive effect. Besides, the block of either TLR2 or TLR4 both reduced IκB phosphorylation. These results above suggest that TLR2-mediated MyD88-dependent pathway contributes to MBP-induced Th1 activation, while TLR4 appears to counteract this effect via MyD88-independent pathway.

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.